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Biotechnology in Agriculture and Forestry

Springer-Verlag Berlin Heidelberg GmbH

Volumes already published


Volume 1: Trees I (1986)
Volume 2: Crops I (1986)
Volume 3: Potato (1987)
Volume 4: Medicinal and Aromatic Plants I (1988)
Volume 5: Trees II (1989)
Volume 6: Crops II (1988)
Volume 7: Medicinal and Aromatic Plants II (1989)
Volume 8: Plant Protoplasts and Genetic Engineering I (1989)
Volume 9: Plant Protoplasts and Genetic Engineering II (1989)
Volume 10: Legumes and Oilseed Crops I (1990)
Volume 11: Somaclonal Variation in Crop Improvement I (1990)
Volume 12: Haploids in Crop Improvement I (1990)
Volume 13: Wheat (1990)
Volume 14: Rice (1991)
Volume 15: Medicinal and Aromatic Plants III (1991)
Volume 16: Trees III (1991)
Volume 17: High-Tech and Micropropagation I (1991)
Volume 18: High-Tech and Micropropagation II (1992)
Volume 19: High-Tech and Micropropagation III (1992)
Volume 20: High-Tech and Micropropagation IV (1992)
Volume 21: Medicinal and Aromatic Plants IV (1993)
Volume 22: Plant Protoplasts and Genetic Engineering III (1993)
Volume 23: Plant Protoplasts and Genetic Engineering IV (1993)
Volume 24: Medicinal and Aromatic Plants V (1993)
Volume 25: Maize (1994)
Volume 26: Medicinal and Aromatic Plants V I (1994)
Volume 27: Somatic Hybridization in Crop Improvement I (1994)
Volume 28: Medicinal and Aromatic Plants VII (1994)
Volume 29: Plant Protoplasts and Genetic Engineering V (1994)
Volume 30: Somatic Embryogenesis and Synthetic Seed I (1995)
Volume 31: Somatic Embryogenesis and Synthetic Seed II (1995)
Volume 32: Cryopreservation of Plant Germplasm I (1995)
Volume 33: Medicinal and Aromatic Plants VIII (1995)
Volume 34: Plant Protoplasts and Genetic Engineering V I (1995)
Volume 35: Trees IV (1996)
Volume 36: Somaclonal Variation in Crop Improvement II (1996)
Volume 37: Medicinal and Aromatic Plants IX (1996)
Volume 38: Plant Protoplasts and Genetic Engineering VII (1996)
Volume 39: High-Tech and Micropropagation V (1997)
Volume 40: High-Tech and Micropropagation V I (1997)
Volume 41: Medicinal and Aromatic Plants X (1998)
Volumes in preparation
Volume 42: Cotton
Volume 43: Medicinal and Aromatic Plants X I

Biotechnology in
Agriculture and Forestry 41
Medicinal and Aromatic Plants X
Edited by Y.P.S. Bajaj

With 228 Figures and 92 Tables

Springer

Professor Dr. Y.P.S. BAJAJ


A-137
New Friends Colony
New Delhi 110065, India

ISSN 0934-943X
ISBN 978-3-642-63748-3
Library of Congress Cataloging-in-Publication Data. Medicinal and aromatic plants. (Biotechnology
in agriculture and forestry; 4-). Includes bibliographies and index. 1. Medicinal plants Biotecnology. 2. Aromatic plants - Biotechnology. 3. Plant cell culture. 4. Materia medica,
Vegetable. I. Bajaj, Y.P.S., 1936- . II. Series. TP248.27.P55M43 1988 660'.62 88-3059.
ISBN 978-3-642-63748-3
ISBN 978-3-642-58833-4 (eBook)
DOI 10.1007/978-3-642-58833-4
This work is subject to copyright. All rights are reserved, whether the whole or part of the material is concerned,
specifically the rights of translation, reprinting, reuse of illustrations, recitation, broadcasting, reproduction on
microfilms or in any other way, and storage in data banks. Duplication of this publication or parts thereof is
permitted only under the provisions of the German Copyright Law of September 9,1965, in its current version,
and permission for use must always be obtained from Springer-Verlag. Violations are liable for prosecution
under the German Copyright Law.
Springer-Verlag Berlin Heidelberg 1998
Originally published by Springer-Verlag Berlin Heidelberg New York 1998
Softcover reprint of the hardcover 1st edition 1998
The use of general descriptive names, registered names, trademarks, etc. in this publication does not imply, even
in the absence of a specific statement, that such names are exempt from the relevant protective laws and
regulations and therefore free for general use.
Cover design: Design & Production, Heidelberg
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SPIN: 10532350

31/3137-5 4 3 2 1 0-Printed on acid-free paper

Dedicated to
Dhanmeet and Paramjit Soin

Preface

This series of books on Biotechnology of Medicinal and Aromatic Plants


provides a survey of the literature focusing on recent information and the
state of the art in tissue culture and the in vitro production of secondary
metabolites. This book, Medicinal and Aromatic Plants X, like the previous
nine volumes published in 1988, 1989, 1991, 1993, 1994, 1995, and 1996, is
unique in its approach. It comprises 22 chapters dealing with the distribution,
importance, conventional propagation, micropropagation, tissue culture
studies, and the in vitro production of important medicinal and other
pharmaceutical compounds in various species of Actinidia, Alkanna, Amebia,
Campanula, Catharanthus, Centella, Chenopodium, Comus, Cynara, Ephedra,
Euglena, Haplophyllum, Morus, Oenothera, Otacanthus, Oxalis, Polypodium,
Rosmarinus, Sesamum, Solanum, Taxus, and Tephrosia. This book is tailored
to the needs of advanced students, teachers, and research scientists in the field
of pharmacy, plant tissue culture, phytochemistry, biochemical engineering,
and plant biotechnology in general.
New Delhi, September 1997

Professor Dr. Y.P.S. BAJAJ


Series Editor

Contents

I Actinidia polygama (Japanese name Matatabi): In Vitro Culture,


Micropropagation, and the Production of Monoterpenes and
Triterpenoids
Y. SHOYAMA, S. CHEN, H. TANAKA, Y. SASAKI, and Y. SASHIDA
(With 5 Figures)
1 Introduction ...............................................
2 In Vitro Culture Studies .....................................
3 Conclusion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

1
5
9
11

II Alkanna tinctoria T. (Alkanets): In Vitro Culture and the


Production of Alkannin and Other Secondary Metabolites
C. GERARDI, G. MITA, E. GRILLO, G. GIOVINAZZO, A. MICELI,
and P. DE LEO (With 9 Figures)
1 Introduction................................................
2 In Vitro Approaches ........................................
3 Conclusion .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

14
17
25
26

III Arnebia euchroma: In Vitro Culture and the Production of


Shikonin and Other Secondary Metabolites
O.V. ZAKHLENJUK and V.A. KUNAKH (With 9 Figures)
1 Introduction ...............................................
2 In Vitro Culture Studies .....................................
3 Summary and Conclusion ....................................
4 Protocol ...................................................
References ............. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

28
30
42
42
43

IV Campanula (Bellflower) Species: In Vitro Culture,


Micropropagation, and the Production of Anthocyanins,
Polyacetylenes, and Other Secondary Metabolites
K. BRANDT and K. ISHIMARU (With 16 Figures)
1 Introduction ...............................................
2 In Vitro Approaches ........................................
3 Conclusion and Prospects ....................................

45
49
62

Contents

4 Protocol
References .................................................. .

63
64

V Catharanthus roseus (Periwinkle): In Vitro Culture,


and High-Level Production of Arbutin by Biotransformation
M. YOKOYAMA and S. INOMATA (With 9 Figures)
1 Introduction................................................
2 Arbutin Production by Biotransformation Using C. roseus
Cell Culture .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
3 Summary ..................................................
4 Prospects ..................................................
5 Protocol ...................................................
References ...................................................

67
69
76
77
77
79

VI Centella asiatica (L.) Urban. (Pennywort): Cell Culture,


Production of Terpeno'ids, and Biotransformation Capacity
J.M. SOLET, A. SIMON-RAMIASA, L. COSSON, and J.L. GUIGNARD
(With 11 Figures)
1 Introduction ...............................................
2 In Vitro Approaches ........................................
3 Summary and Conclusion ....................................
References .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

81
85
93
93

VII Chenopodium album L. (Fat Hen): In Vitro Cell Culture,


and Production of Secondary Metabolites (Phytosterols and
Ecdysteroids)
M.-F. CORIO-COSTET, L. CHAPUIS, and J.-P. DELBEcQuE
(With 7 Figures)
1 Introduction................................................
2 In Vitro Production of Sterols and Steroids ....................
3 Summary and Conclusion ....................................
4 Protocols ..................................................
References. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

97
99
107
110
110

VIII Comus kousa (Dogwood): In Vitro Culture, and the


Production of Tannins and Other Phenolic Compounds
(With 14 Figures)
K. ISHIMARu, N. TANAKA, T. KAMIYA, T. SATO, and K. SHIMOMURA
(With 14 Figures)
1 Introduction ...............................................
2 Tannin Constituents in Comus Plants. . . . . . . . . . . . . . . . . . . . . . . . . .
3 In Vitro Approaches ........................................
4 Summary and Conclusion ....................................
5 Protocol ...................................................
References ..................................................

113
114
115
128
128
129

Contents

XI

IX Cynara cardunculus subsp. flavescens (Cardoon): In Vitro Culture,


and the Production of Cyprosins - Milk Clotting Enzymes
M.e. CORDEIRO, M.S. PAIS, and P.E. BRODELIUS (With 10 Figures)
1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
2 In Vitro Culture Studies. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
3 Isolation of the Milk-Clotting Enzymes ........................
4 Characterization of the Enzymes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
5 Primary Structure of Cyprosin ................................
6 Modeling the Structure of Cyprosin ...........................
7 Tissue-Specific Accumulation of Cyprosin .. . . . . . . . . . . . . . . . . . . . .
8 Localization of Cyprosins in the Flower Tissues ....... . . . . . . . . . .
9 Possible Physiological Functions of Cyprosins . . . . . . . . . . . . . . . . . . .
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

132
135
138
138
142
145
146
148
150
151

X Ephedra Species: In Vitro Culture, Micropropagation and the


Production of Ephedrine and Other Alkaloids
N.A O'DOWD, P.G. MCCAULEY, G. WILSON, l.AN. PARNELL,
T.A.K. KAVANAGH, and D.l. MCCONNELL (With 15 Figures)
1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
2 Distribution of the Ephedrines . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
3 In Vitro Culture Studies .....................................
4 Summary and Conclusion ....................................
References. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

154
157
167
187
189

XI Euglena gracilis Z: Biotransformation of Terpenoids and


Related Compounds
Y.NOMA and Y. ASAKAWA (With 22 Figures)
1 Introduction ...............................................
2 Cultivation........ . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
3 Biotransformation of Terpenoids and Related Compounds .......
4 Summary and Conclusion ....................................
References ..... . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

194
195
195
233
236

XII Haplophyllum patavinum (L.) G. Don fil. (Paduan rue): In Vitro


Regeneration, and the Production of Coumarin Compounds
E.M. CAPPELLETTI, G. INNOCENTI, R. CANIATO, R. FILIPPINI,
and A PIOVAN (With 7 Figures)
1 Introduction ...............................................
2 In Vitro Approaches. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
3 Conclusion ................................................
References .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

238
241
257
257

XII

Contents

XIII Marus Species (Mulberry): In Vitro Culture, Micropropagation,


and the Formation of Mulberrofuran, Kuwanol, and Other
Secondary Metabolites
Y.P.S. BAJAJ, J. IVANH~KA, and S. UEDA (With 14 Figures)
1
2
3
4
5

General Account. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
In Vitro Culture Studies .....................................
Micropropagation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
In Vitro Production of Secondary Metabolites in Marus alba .....
Extraction and Structure of Intermolecular Diels-Alder-Type
Adducts of Prenylchalcone and Prenylated 2-Arylbenzofuran .....
6 Summary and Conclusion ....................................
7 Protocol for Micropropagation ...............................
References ..................................................

261
264
265
271
279
281
281
282

XIV Oenathera Species (Evening Primrose): In Vitro Regeneration,


Production of Flavonoids, Fatty Acids, and Other Secondary
Metabolites
L. SKRZYPCZAK, B. THIEM, and M. WESrn.OWSKA
(With 5 Figures)
1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
2 Compounds in Oenathera Species .............................
3 In Vitro Approaches ........................................
4 Conclusion .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
5 Protocol ...................................................
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

286
288
293
299
300
300

XV Otacanthus Species: In Vitro Culture, Plant Propagation,


and the Production of Essential Oil
A.c. RONSE, H. DE POOTER, A. VAN DE VYVER
and M.P. DE PROFT (With 7 Figures)
1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
2 Volatile Constituents of the Intact Plant .......................
3 In Vitro Culture Studies .....................................
4 Conclusion and Prospects ....................................
References. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

305
307
308
318
319

XVI Oxalis Species: In Vitro Culture, Micropropagation, and the


Formation of Anthocyanins
J. VAN STADEN (With 5 Figures)
1 Introduction................................................
2 In Vitro Culture Studies .....................................
3 Anthocyanin Production .....................................
4 Protocol ...................................................
References ... . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

320
322
329
330
332

Contents

XIII

XVII Polypodium vulgare L. (Wood Fern): In Vitro Cultures and the


Production of Phytoecdysteroids
J. MESSEGUER, E. MEL., N. REIXACH, J. IRRURE-SANTILARI,
and J. CASAS (With 9 Figures)
1
2
3
4
5
6

Introduction ...............................................
In Vitro Culture Studies .....................................
Phytoecdysteroid Content in In Vitro Cultures . . . . . . . . . . . . . . . . . .
Biosynthetic Studies of Ecdysteroids in In Vitro Cultures ........
Summary and Conclusion ....................................
Protocol for Extraction and Quantification of Ecdysteroids in
P. vulgare . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

333
335
338
341
344
345
346

XVIII Rosmarinus officinalis L. (Rosemary): In Vitro Culture,


Regeneration of Plants, and the Level of Essential Oil and
Monoterpenoid Constituents
A.A. TAWFIK, P.E. READ, and S.L. CUPPETT (With 9 Figures)
1 General Account ...........................................
2 In Vitro Culture Studies .....................................
3 Effect of Medium Composition on Growth and Monoterpene
Levels. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
4 Summary ..................................................
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

349
353
355
363
364

XIX Sesamum indicum L. (Sesame): In Vitro Culture, and the


Production of Naphthoquinone and Other Secondary Metabolites
T. OGASAWARA, K. CHIBA, and M. TADA (With 8 Figures)
1 Introduction................................................
2 In Vitro Approaches ........................................
3 Summary ..................................................
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

366
373
389
389

XX Solanum mammosum L. (Terong Susu): In Vitro Culture


and the Production of Steroidal Alkaloids and Other Secondary
Metabolites
G. INDRAYANTO, R. SONDAKH, A. SYAHRANI, and W. UTAMI
(With 12 Figures)
1 Introduction................................................
2 In Vitro Culture Studies .....................................
3 Conclusion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
4 Protocols for Tissue Culture . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

394
399
409
409
411

XIV

Contents

XXI Taxus Species (Yew): In Vitro Culture, and the Production


of Taxol and Other Secondary Metabolites
E.R.M. WICKREMESINHE and R.N. ARTECA (With 16 Figures)
1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
2 In Vitro Approaches ........................................
3 Conclusion and Prospects ....................................
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

415
419
437
438

XXII Tephrosia vogelii Hook f.: In Vitro Culture, and the


Production of Rotenoids and Other Secondary Metabolites
N. LAMBERT, M.-F. TROUSLOT, and H. CHRESTIN (With 8 Figures)
1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
2 In Vitro Approaches ........................................
3 Conclusions ................................................
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

443
446
453
454

Subject Index. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

457

List of Contributors

ARTECA, R.N., Department of Horticulture, The Pennsylvania State


University, University Park, Pennsylvania 16802, USA
ASAKA W A, Y., Faculty of Pharmaceutical Sciences, Tokushima Bunri
University, Yamashiro-cho, Tokushima 770, Japan
BAJAJ, Y.P.S., A-137 New Friends Colony, New Delhi 110065, India
BRANDT, K., Research Group for Plant Breeding and Propagation,
Department of Ornamentals, Danish Institute of Plant and Soil Science,
Kirstinebjergvej 10, DK-5792 Arslev, Denmark
BRODELIUS, P.E., Department of Plant Biochemistry, Lund University,
P.O.Box 117, S-22100 Lund, Sweden
CANIATO, R., Department of Biology, University of Padua, Via U. Bassi
58/13, 35131 Padua, Italy
CAPPELLETTI, E.M., Department of Biology, University of Padua, Via U.
Bassi 58/13, 35131 Padua, Italy
CASAS, J., Department of Biological Organic Chemistry, CID (CSIC), J.
Girona, 18, 08034 Barcelona, Spain
CHAPUIS, L., INRA-Bordeaux, URIV-Phytopharmacie, BP 81, 33883
Villenave d'Ornon, France
CHEN, S., Faculty of Pharmacutical Sciences, Kyushu University, 3-1-1
Maidashi, Higashi-ku, Fukuoka 812, Japan
CHIBA, K., Laboratory of Bio-organic Chemistry, Tokyo University of
Agriculture and Technology, Saiwai-cho, Fuchu-shi, Tokyo 183, Japan
CHRESTIN, H., Laboratoire de Physiologie et Biotechnologie Vegetales,
ORSTOM-IIRSDA, BP V51, Abidjan, Cote d'Ivoire

XVI

List of Contributors

COSSON, L., Laboratoire de Botanique, Faculte de Pharmacie,


Universite Paris-Sud, 5, rue JB Clement, 92296 Chatenay-Malabry
Cedex, France
CORDEIRO, M.e., Department of Plant Biochemistry, Lund University,
P.O.Box 117, S-22100 Lund, Sweden
CORIO-COSTET, M.F., INRA-Bordeaux, URIV-Phytopharmacie, BP 81, 33883
Villenave d'Ornon, France
CUPPETT, S.L., Department of Food Sciences and Technology, University of
Nebraska, Lincoln, Nebraska 68583-0919, USA
DELBECQUE, J.P., Universite de CNRS UMR 5548 (Developpement, Communication Chimique), Bourgogne 6 Bd Gabriel, 21000 Dijon, France
DE LEO, P., Dipartimento di Fisiologia Vegetale, Universita di Leece, Via
Monteroni, 73100 Leece, Italy
DE POOTER, H., Formerly: Laboratory for Organic Chemistry, Faculty of
Agronomy, University of Gent, Coupure Links 653, B-9000 Gent, Belgium
DE PROFT, M.P., Laboratory for Plant Physiology, Faculty of Agronomy,
Catholic University of Leuven, De Croylaan 42, B-3001 Leuven, Belgium
FILIPPINI, R., Department of Biology, University of Padua, Via U. Bassi
58/13, 35131 Padua, Italy
GERARDI, e., Istituto di Ricerca sulle Biotecnologie Agroalimentari, CNR,
Via Monteroni, 73100 Leece, Italy
GIOVINAZZO, G., Istituto di Ricerca sulle Biotecnologie Agroalimentari,
CNR, Via Monteroni, 73100 Leece, Italy
GRILLO, G., Dipartimento di Fisiologia Vegetale, Universita di Leece, Via
Monteroni, 73100 Leece, Italy
GUIGNARD, J.L., Laboratoire de Botanique, Faculte de Pharmacie,
Universite Paris-Sud, 5, rue JB Clement, 92296 Chatenay-Malabry Cedex,
France
INDRAYANTO, G., Laboratory of Pharmaceutical Biotechnology, Faculty of
Pharmacy, Airlangga University, J1. Dharmawangsa dalam, Surabaya 60286,
Indonesia
INNOCENTI, G., Department of Pharmaceutical Sciences, University of
Padua, Via Marzolo 5, 35123 Padua, Italy

List of Contributors

XVII

INOMATA, S., Pharmaco Science Research Laboratories, Shiseido


Research Center, 1050 Nippa-cho, Kohoku-ku, Yokohama 223, Japan
IRURRE-SANTILARI, J., Department of Biological Organic Chemistry, CID
(CSIC), J. Girona, 18,08034 Barcelona, Spain
ISHIMARU, K., Department of Applied Biological Sciences, Faculty of
Agriculture, Saga University, 1 Honjo, Saga 840, Japan
IVANICKA, J., Centre of Development of Horticulture, Hornonitrianska 20,
971-01 Prievidza, Slovakia
KAMIYA, T., Chichibu Onoda Cement Corporation, Tsukuba Biotechnology
R&D Center, 25-13, l-Chome Kannondai, Tsukuba, Ibaraki 305, Japan
KAVANAGH, T.AK., Department of Genetics, University of Dublin, Trinity
College, Dublin 2, Ireland
KUNAKH, V.A Institute of Molecular Biology and Genetics, National
Academy of Sciences of Ukraine, Kiev 252143, Ukraine
LAMBERT, N., Laboratoire de Physiologie et Biotechnologie Vegetales,
ORSTOM-IIRSDA, BP V51, Abidjan, Cote d'Ivoire Present address:
Analytical Product Division, Millipore S.A BP 307, 78054 Saint Quentinen-Yvelines Cedex, France
MCCAULEY, P.G., School of Botany, University of Dublin, Trinity College,
Dublin 2, Ireland
MCCONNELL, D.J., Department of Genetics, University of Dublin, Trinity
College, Dublin 2, Ireland
MELE, E., Department of Plant Genetics, IRTA, Centre de Cabrils, Crta. de
Cabrils sin, 08348 Cabrils, Spain
MESSEGUER, J., Department of Plant Genetics, IRTA, Centre de Cabrils,
Crta. de Cabrils sin, 08348 Cabrils, Spain
MICELI, A, Dipartimento di Fisiologia Vegetale, Universita die Lecce, Via
Monteroni, 73100 Lecce, Italy
MITA, G., Istituto di Ricerca sulle Biotechnologie Agroalimentari, CNR, Via
Monteroni, 73100 Lecce, Italy
NOMA, Y., Faculty of Domestic Sciences, Tokushima Bunri University,
Yamashiro-cho, Tokushima 770, Japan

XVIII

List of Contributors

O'DOWD, N.A, TEAGASC, Food and Agriculture Development Authority,


Kinsealy Research and Development Centre, Malahide Road, Dublin 17,
Ireland
OGASAWARA, T., Research Institute of Q.P. Corporation, 5-13-1, Sumiyoshicho, Fuchu-shi, Tokyo 183, Japan
PAIS, M.S., Centro de Biotecnologia Vegetal, Faculdade de Ciencias de
Lisboa, Bloca C2, Campo Grande, P-1700 Lisboa, Portugal
PARNELL, J.AN., School of Botany, University of Dublin, Trinity College,
Dublin 2, Ireland
ProVAN, A, Department of Pharmaceutical Sciences, University of Padua,
Via Marzolo 5, 35123 Padua, Italy
READ, P.E., Horticulture Department, University of Nebraska Lincoln,
Nebraska 68583-0724, USA
REIXACH, N., Department of Biological Organic Chemistry, CID (CSIC),
J. Girona, 18, 08034 Barcelona, Spain
RONSE, AC., National Botanic Garden, Domein van Boechout, B-1860
Meise, Belgium
SASAKI, Y., Oita Prefectural Forest Experiment Station, Hita Oita, 877-13,
Japan
SASHIDA, Y., Tokyo College of Pharmacy, 1432-1 Horinouchi, Tokyo
1992-03, Japan
SATO, T., Chichibu Onoda Cement Corporation, Tsukuba Biotechnology
R&D Center, 25-13, 1-Chome Kannondai, Tsukuba, Ibaraki 305, Japan
SHIMOMURA, K., Tsukuba Medicinal Plant Research Station, National
Institute of Health Sciences, 1 Hachimandai, Tsukuba, Ibaraki 305, Japan
SHOYAMA, Y., Faculty of Pharmaceutical Sciences, Kyushu University, 3-1-1
Maidashi, Higashi-ku, Fukuoka 812, Japan
SIMON-RAMIASA, A, Laboratoire de Botanique, Faculte de Pharmacie,
Universite Paris-Sud, 5, rue JB Clement, 92296 Chatenay-Malabry Cedex,
France
SKRZYPCZAK, L., Department of Pharmaceutical Botany, K. Marcinkowski
University of Medical Sciences in Poznan, Wieniawskiego 1, 61-712 Poznan,
Poland

List of Contributors

XIX

SOLET, J.M., Laboratoire de Botanique, Faculte de Pharmacie, Universite


Paris-Sud, 5, rue JB Clement, 92296 Chiitenay-Malabry Cedex, France
SONDAKH, R, Laboratory of Pharmaceutical Biotechnology, Faculty of
Pharmacy, Airlangga University, J1. Dharmawangsa dalam, Surabaya 60286,
Indonesia
SYAHRANI, A, Laboratory of Pharmaceutical Biotechnology, Faculty of
Pharmacy, Airlangga University, J1. Dharmawangsa dalam, Surabaya 60286,
Indonesia
TADA, M., Laboratory of Bio-organic Chemistry, Tokyo University of
Agriculture and Technology, Saiwai-cho, Fuchu-shi, Tokyo 183, Japan
TANAKA, H., Faculty of Pharmaceutical Sciences, Kyushu University, 3-1-1
Maidashi, Higashi-ku, Fukuoka 812, Japan
TANAKA, N., Chichibu Onoda Cement Corporation, Tsukuba Biotechnology
R&D Center, 25-13, l-Chome Kannondai, Tsukuba, Ibaraki 305, Japan
TAWFIK, AA, Horticulture Department, College of Agriculture, Assiut
University, Assiut 71526, Egypt
THIEM, B., Department of Pharmaceutical Botany, K. Marcinkowski
University of Medical Sciences in Poznan, Wieniawskiego 1,61-712 Poznan,
Poland
TRoUSLOT, M.-F., Laboratoire de Ressources Genetiques et Amelioration
des PI antes Tropicales, ORSTOM, BP 5045, 34032 Montpellier, France
UEDA, S., Formerly: Faculty of Pharmaceutical Sciences, Kyoto University,
Sakyo-ku, Kyoto 606-01, Japan
UTAMI, W., Laboratory of Pharmaceutical Biotechnology, Faculty of
Pharmacy, Airlangga University, J1. Dharmawangsa dalam, Surabaya 60286,
Indonesia
VAN DE VYVER, AM., National Botanic Garden, Domein van Boechout,
B-1860 Meise, Belgium
VAN STADEN, J., Natal University Research Unit for Plant Growth and
Development, Department of Botany, University of Natal Pietermaritzburg,
Private Bag X01, Scottsville 3209, South Africa
WESOWWSKA, M., Department of Pharmaceutical Botany, K. Marcinkowski
University of Medical Sciences in Poznan, Wieniawskiego 1, 61-712 Poznan,
Poland

xx

List of Contributors

WICKREMESINHE, E.R.M., Department of Horticulture, The Pennsylvania


State University, University Park, Pennsylvania 16802, USA
WILSON, G., School of Botany, University College Dublin, Belfield, Dublin
4, Ireland
YOKOYAMA, M., Pharmaco Science Research Laboratories, Shiseido
Research Center, 1050 Nippa-cho, Kohoku-ku, Yokohama 223, Japan
ZAKHLENJUK, O.V., Institute of Molecular Biology and Genetics, National
Academy of Sciences of Ukraine, Kiev 252143, Ukraine

I Actinidia polygama (Japanese name Matatabi): In


Vitro Culture, Micropropagation, and the Production
of Monoterpenes and Triterpenoids

1 Introduction
1.1 Botanical Aspects

The genus Actinidia (family Actinidiaceae) consists of 40 species found in


tropical and subtropical Asian countries. Actinidia species have been utilized
for medicinal purposes and as a food source. For instance, the sour fruit of A.
chinensis is very rich in vitamin C. The roots and racemes of A. chinensis are
utilized as an astringent, to quench thirst, and as a diuretic. The fruits and
leaves of A. arguta are considered to be antipyretic, astringent, tonic, thirstquenching, and insecticidal. The roots and leaves of A. eriantha are utilized as
an antidote, with antipyretic effect. The fruits of A. coriacea are also utilized as
an antipyretic.
Actinidia polygama is indigenous in Japan and Korea (Fig. 1). It is a
deciduous shrub which grows on the edge of streams. The stem grows to
several meters in length and 5 cm in diameter. The leaf is egg-shaped and
alternately arranged. The leaf surface frequently changes to a white color in
and around June. The shrub produces male and female white flowers in early
summer. The fruits frequently form galls, which are induced by cecidogenous
midges, Asphondylia matatabi.
1.2 Biologically Active Compounds and Traditional Uses

The fruit galls of A. polygama are commonly known as mu tian liao in


Chinese traditional medicine. It has also been used as an analgesic, a tonic,
an anti-rheumatic and digestive in folk medicine in Japan. However, the
active principles have not yet been determined. The volatile components
which cause the well-known specific activity of Felidae species were
reported (Hazama 1942); cats show an especial liking for this plant, first
licking and then eating it. Generally, they salivate, rub their fur onto the

I Faculty of Pharmaceutical Sciences, Kyushu University, 3-1-1 Maidashi, Higashi-ku, Fukuoka


812,Japan
2 Oita Prefectural Forest Experiment Station, Hita Oita 877-13, Japan
3 Tokyo College of Pharmacy, 1432-1 Horinouchi, Tokyo 192-03, Japan

Biotechnology in Agriculture and Forestry. Vol. 41


Medicinal and Aromatic Plants X (ed. by Y.P.S. 8ajaj)
Springer-Verlag Berlin Heidelberg 1998

Y. Shoyama et al.

Fig. 1. Actinida polygama plant.

fruit gall, and finally sleep. The fruit galls contain cyclopentanoid
monoterpenes like iridomyrmecin, which is the most active stimulant
for cats (Sakan et al. 1960). In 1949, Pavan reported that iridomyrmecin
isolated from the anal glands of the Argentine ant, Iridomymex humilis,
possessed insecticidal activity. The workers of 1. humilis use this secretion
for attack and defense against their insect enemies, and presumably this
ability to wage chemical warfare is one of the factors responsible for the
expansion of the species. The occurrence of similar defensive substances
which may protect the plant against phytophagous insects has been noted.
Sakan et al. studied the plant and isolated a number of other monoterpenoids.
Figure 2 shows the biologically active monoterpenoids of A. polygama. They
consist of four groups, aldehydes (iridodial, dehydroiridodial), alcohols
(iridodiol, dehydroiridodiol, and 5-hydroxyiridodiol: strongly attractive
for Chyrysopa septempunctata and C. japana), lactones (iridomyrmecine,
dihydronepetalactone, nepetalactone, neonep etalactone: attractive to cats),
and an artificial compound (actinidine: attractive to cats). Alcohols
are specifically attractive to Felidae species. Aldehydes were reported to
be a bitter principle. It is most remarkable that cyclopentanoid monoterpenes were found to strongly attract only the male adults of Chrysopa
septempunctata (lacewing, stink flies) and C. japana in the amount of 1O- 6!lg of
neomatatabiol and isoneomatatabiol and 1O-3!lg of dehydroiridodiol, as
indicated in Table 1.
In addition, the fruit galls of A. polygama contain several triterpenoids
(Sashida et al. 1992). Some types of terpenoids have long been recognized
as carriers of important physiological functions and they are also substances
of vital importance for living organisms, i.e., some steroids, carotenoids,

Actinidia polygama (Japanese name Matatabi)

ecdysone, and, in plants, phytol, which is an essential component of the chlorophyll molecule. Furthermore, triterpenoids constitute one of the largest
groups with highly diversified biological activities including antiinflammatory,
antineoplast, antibacterial, antifungal activities, diuretic, antidiabetic, and

R1
iridodiol
iridodial

R2

CH 2 0H CH 2 0H
CHO
CHO

actlnidine

5-hydroxymatatabiether

~
-;/'

matatabiol

dehydroindodiol
dehydroiridodial

HO ,I,

7-hydroxymatatabiethe r

cQ
OH

[~
ailomatatabibiol

R1

neomatatablol
isoneomatatabiol

R2

R1

R2

H
CH 3

CH 3
H

Fig. 2A,B. Structures of biologically active monoterpenoids from Actinidia polygama. (Sakan
et al. 1964)

Y. Shoyama et al.

nepetalactone

iridomyrmecin
B isoiridomyrmecin

Rl
H
CH3

neonepetalactone

dihydronepetalactone
isodihydronepetalactone

Rl
H
CH3

Fig. 2A,B. Continued

Table 1. Potent cyclopentanoid monoterpenes isolated from


Actinidia polygama attractive to male adults of Chrysopa
septempunctata and C. japana. (Sakan et al. 1964)

Compound
Iridodiol
5-H ydroxymatatabiether
7-Hydroxymatatabiether
Allomatatabiol
Matatabiol
Dehydroiridodiol
Neomatatabiol
Isoneomatatabiol

Active amount
(ug)
1
1
1
1

10- 3
10- 3
10- 6
10- 6

metabolite-displacing activity (Kapoor and Chawla 1986; Padmaja et al. 1993).


Inhibitory effects of triterpenoids and their saponins on skin tumor promotion
and Epstein-Barr virus activation have been studied by Konoshima et al.
(1987, 1992, 1994). In view of these facts, the triterpenoids of A. polygama
were studied.

Actinidia polygama (Japanese name Matatabi)

2 In Vitro Culture Studies


Although numerous studies have been conducted on various in vitro
aspects (Huang et al. 1988) of Actinidia chinensis and A. deliciosa, i.e.,
micropropagation (Monette 1986), protoplast culture (Cai et al. 1993),
genetic transformation (Oliveira et al. 1994), conservation of germplasm
(Monette 1995), etc., no studies exist on Actinidia polygama except our work
(Shoyama et al. 1989; Sashida et al. 1992, 1994), which is summarized in this
chapter.
2.1 Callus Formation from Fruit Galls

2.1.1 Materials and Methods

Fruit galls of A. polygama were collected from native strains at Sagamiko


in Kanagawa Prefecture in Japan. The natural plants, stems, leaves, and
roots were collected at Oume in Tokyo. The fruit galls were washed with
tap water, and sterilized with 1% NaOCI for 10min, then with 70% EtOH
for 30 s, and finally washed twice thoroughly with sterilized water. The
pericarps were removed, and the sarcocarp dissected into a cube (3mm 3).
The basal medium consisted of BS (Gamborg et al. 1968), LS (Linsmaier
and Skoog 1965), and MS (Murashige and Skoog 1962) salt with (in
mg/l): myoinositol, 100; nicotinic acid, O.S; pyridoxine HCI, O.S; thiamine
HCI, 0.1; glycine, 2; sucrose, 30000; agar, 9000, supplemented with auxins
(IBA, NAA), BAP, kinetin, and GA. All culture tubes contained 30ml
of medium adjusted to pH S.S before autoclaving. The cultures were
exposed to 16h light, 2000-2S00lx, from cool white fluorescent tubes at
2S 1C.
At the initiation stage of callus induction, the peri carp segments were
cultured for 30 days. In the second stage, callus induced was subcultured for 30
days. Shoot primordia were separated from the callus and transferred to
shoot-forming medium and cultured for 30 days. The regenerated shoots were
subcultured on the root-forming medium for 30 days.
2.1.2 Induction of Callus from Fruit Galls

Callus was induced from fruit galls on the BS medium supplemented with
NAA, BAP, and kinetin (1 mg/l each) or on the LS medium containing lOmg/
I NAA and 1 mg/l kinetin. Callus growth was better on the BS than on LS
medium, as indicated in Table 2. When callus was subcultured on the MS
medium containing IBA, growth was favored. The addition of 3 mg/l IBA
favored callus growth better than the addition of a higher concentration
(Table 2), thus this medium was routinely used in the callus propagation
stage (Fig. 3a).

Y. Shoyama et al.

Fig. 3a-d. Propagation system of Actinidia polygama by callus culture (Y. Shoyama et al..
unpubl.). a Callus culture induced from fruit gall segment on MS medium supplemented
with IBA (3mg/I). b Formation of shoot primordia-like bud after three subcultures on BS
medium supplemented with NAA, BAP, and kinetin (1 mg/I each). c Shoot formation on
LS medium supplemented with 2,4-D (O.S mg/I each). d Plantlet formation on hormone-free MS
medium

Table 2. Propagation of callus induced from fruit gall of A. polygama on different media.
(Shoyama et al. 1989; Sashida et al. 1994)
Basal media
Hormone
(mg 1-1)
Fresh weight
(mg culture -1)

Gamborg BS

Linsmaier-Skoog

NAA-BAP-Kin
(1:1:1)

NAA-Kin
(10:1)

730

240

Murashige-Skoog

270

IBA
3

970

390

Culture conditions: 2S :+: 1C, 16h light, 1 month. NAA: I-naphthaleneacetic acid, BAP:
benzylaminopurine, Kin: kinetin, IBA: indol-3-butyric acid.

Actinidia polygama (Japanese name Matatabi)

2.1.3 Differentiation from Callus

When the callus induced by the medium supplemented with NAA, BAP,
and kinetin (1 mg/l each) was subcultured on the same medium for three
generations, shoot primordia and adventitious roots appeared on its surface
(Fig. 3b). Some of the shoot buds were transferred to LS medium supplemented with 2,4-D and BAP (0.5 mg/l each), resulting in shoot and root
regeneration (Fig. 3c). Shoots were subcultured on MS medium supplemented
with NAA (1 mg/l) , or on hormone-free medium to induce root formation
(Fig. 3d).
Chromosome numbers (2n = 58) in the root tip of the plantlet regenerated
from callus were the same as in the mother plants (Kitamura and Murata
1979).
2.2 Micropropagation

Shoots of regenerated plantlets on hormone-free MS medium were cut into


stem segments having one leaf. The segments were cultured on MS medium
supplemented with BAP (0.1, 0.5, 1, or 2mg/l) for 1 month. The elongated
shoots were cut into segments and subcultured on the same medium for 1
month. Stem segments were cultured on hormone-free MS medium for 1
month to produce plantlets, which were transferred to vermiculite and cultured for 3 months.
Shoots differentiated from the callus were transferred to hormone-free
MS medium, resulting in plantlets, as indicated in Fig. 3d.
Regenerated shoots were cultured on MS medium supplemented with
various concentration of BAP (0.1, 0.5, 1, or 2.5mg/l). Only lower BAP concentration (0.1 mg/l) was favorable for shoot elongation. The stem of an elongated shoot was cut into six segments each having one leaf. The stem segment
regenerated shoots perfectly on hormone-free MS medium, resulting in
plantlets (Fig. 3d). Since a single shoot propagates six times in 1 month, the
micropropagation system can be used routinely. Young plants were transferred to vermiculite and cultivated for 3 months. The transplantation was
perfect, the plantlets growing to 30cm in height after 6 months of culture in
vermiculite.
2.3 Productiou of Mouoterpeues aud Triterpeuoids iu Callus Culture aud
Regeuerated Plautlet (Shoyama et al. 1989; Sashida et al. 1994)
2.3.1 Materials and Methods

The fresh callus propagated on MS medium containing lEA (3 mg/l) was


extracted with MeOH. The MeOH solution was evaporated in vacuo. The
extract was reextracted with ethylether. This crude terpenoid fraction was
subjected to GC-MS.

Y. Shoyama et al.

The callus was extracted with hot EtOH several times, and the EtOH
solution was evaporated in vacuo. The extract was suspended in H 2 0.
The suspension was extracted with EtOAc and BuOH, successively. The
EtOAc soluble part was repeatedly chromatographed on a silica gel column
(CHCI3-Me2CO and CHCI3-EtOAc system) to distinguish individual
triterpenoids.
Structures were determined for these triterpenoids for their lR, ElMS,
FABMS, lH_, and 13C-NMR data (Cheung and Tokes 1968; Sakakibara and
Kaiya 1983; Kikuchi et al. 1984; Kojima and Ogura 1986; Kojima et al. 1987;
Bhandari et al. 1990; Sashida et al. 1994).

2.3.2 Results
Figure 4 shows the GC-MS spectrum of the crude terpenoid fraction
obtained from callus culture. Although the GC spectrum indicated that many
volatile compounds were contained in the crude terpenoid fraction, only
dihyronepetalactone (or isodihydronepetalactone), which is a major constituent (Fig. 4, arrow), was determined. The stereochemistry of this compound is
still unknown.
Figure 5 shows the structure of triterpenoids isolated from callus culture
and fruit gall. Table 3 shows the distribution of triterpenoids in callus tissue,
regenerated plantlets, in vivo plants, and fruit galls reported previously
(Sashida et al. 1992). It became clear that the triterpenoid contents in both in
vivo and regenerated plants are the same, in agreement with the fact that they
have the same chromosome number (2n = 58) (Kitamura and Murata 1979),
indicating that no variation occurred in callus culture. 3j3,24-dihydroxyurs12-en-28-oic acid and 2a,3j3,24-trihydroxyurs-12-en-28-oic acid are found only
in callus tissue, and this is the first example isolated from natural sources.
3j3,24-dihydroxyurs-12-en-28-oic acid may be an important product in the
oxidation process between delta-12 and delta-ll triterpenoid, because
hydroxylation of C-13 may occur through the epoxide on C-12 and C-13.
This introduction of a hydroxyl group on C-12 of triterpenoids is variable
for the transformation of various triterpenoids. 2a,3j3,24-trihydroxyurs-11-en13j3,28-0Iide, 3j3,24-dihydroxyurs-12-en-28-oic acid, 2a,3a,24-trihydroxyurs12-en-28-oic acid, 2a,3j3,24-trihydroxyurs-12-en-28-oic acid, and 3j3-( transp-coumaroyloxy)-2a,24-dihydroxyurs-12-en-28-oic acid, which are contained
in callus tissue, have the 4-j3-CH2 0H group. On the other hand, 2a,3a,23trihydroxyurs-12-en -28-oic acid, 2a,3j3 ,23-trihydroxyurs-12-en -28-oic acid,
and 3j3-(trans-p-coumaroyloxy)-2a,23-dihydroxyurs-12-en-28-oic acid having
the 4-CH2 0H group are not found in callus tissue. This clearly shows that
the hydroxylation ability of the C-4 dimethyl group in natural plants, regenerated plants, and fruit galls is nonspecific, but that in callus tissue is
specific. Therefore, it is speculated that the selective hydroxylation ability
of callus tissue can be used for the selective biotransformation of the
cyclohexane ring having the C-4 dimethyl group. The result that fruit galls
indicated the intermediate triterpenoid pattern between the in vivo plants

Actinidia polygama (Japanese name Matatabi)

GC-MS
c/o OV-17(1.1 m x 2.6 mm),

He: 40 ml/min, 152C

15

10

min

0
250C

II

Ii

50

III

70eV

I
.1

I.

1111

1.11
'

iI

15(1

'

iIi

170

Fig. 4. Analysis of monoterpenoids by gas chromatography-mass spectrometry. (Shoyama et al.


1989). GC-MS spectrometry was carried out by the Shimadzu OP-1000. GC and MS conditions
were indicated in individual spectrum, respectively. Peak indicated by arrow analyzed by
MS spectrometry in MS spectrum indicating that the molecular peak was mle 168 in the MS
spectrum

and callus tissue shows that fruit galls may possess greater metabolic ability
than callus tissue.

3 Conclusion
The procedure described here can be used as a simple method of
micropropagation, thereby helping to produce rapid strains of A. polygama.
Moreover, since the chromosome numbers of A. polygama in the root tip of
plantlets regenerated by callus culture were the same as those of the mother
plants (Kitamura and Murata 1979), this system can be utilized to supply a
homogeneous population of this plant. Although the sex of regenerated
plantlets has not been determined, it may be possible to propagate either
female or male plantlets by this system.

R4
24

727 15

'0

""

""

a-OH
CHzOH CH 3
!3-0H
CHzOH CH 3
!3 ~p--Coumaroyloxy CHzOH CH 3

OH
OH
OH

R4

CH 3
!3~OAc
CH 3
!3-0H
CH 3
!3-0H
CH 3
ex-OH
CH 3
!3-0H
CH 3
!3 -p--Coumaroyloxy CH 3

CH 3
CH 3
CH 3
CHzOH
CHzOH
CHzOH
CHzOH

!3~OH

OH
H
OH
H
OH
OH
OH

2 ex, 3 ex,

Rz
R3

"

HO""

HO"

R,

22

21

24~trihydroxyurs~

OH

--;/'

11 ~en~ 13 !3,

28~olide

Fig. 5. Structures of triterpenoids isolated from Actinidia polygama callus. (Reprinted from Sashida et a!., copyright 1994, Phytochemistry 35:377-380,
with kind permission from Elsevier Sciences Ltd, The Boulevard, Langford Lane, Kidlington OX5 1GB, UK)

3~O-acetylursolic acid
2 ex, :l!3 ~dihydroxyurs~ 1 2~en~28~oic acid
313, 24-dihydroxyurs-12-en-28-oic acid
2 ex, 3 IX, 24-trihydroyurs-12-en-28-oic acid
2 IX, 313, 24-trihydroyurs~ 12-en-28-oic acid
313 - (trans-p--coumaroyloxy) -2 ex, 24-dihydroxyurs-12-en-28-oic acid
2 a, 3 a, 23-trihydroyurs-12-en-28-oic acid
2 a, 3!3, 23-trihydroyurs-12~en~28-oic acid
3/3- (trans-p--coumaroyloxy) ~2 IX, 23-dihydroxy~
urs~ 12~en~28-olc acid

ursolic acid

25

:: 20

30

'a"
'~"

'<

::r

VJ

!-<

>-'

Actinidia polygama (Japanese name Matatabi)

11

Table 3. Distribution of the triterpenoids in callus, regenerated plants, in vivo plants, and galls of
A. polygama. (Sashida et al. 1994)

Compound
Ursolic acid
3-0-acetylursolic acid
2a, 3j3-dihydroxyurs-12-en-28-oic acid
2a, 3a, 24-trihydroxyurs-ll-en-13 {3, 28-olide
3{3, 24-dihydroxyurs-12-en-28-oic acid
2a, 3a, 24-trihydroyurs-12-en-28-oic acid
2a, 3{3, 24-trihydroyurs-12-en-28-oic acid
3{3-(trans-p-coumaroyloxy)-2a, 24-dihydroxy
-urs-12-en-28-oic acid
2a, 3a, 23-trihydroyurs-12-en-28-oic acid
2a, 313, 23-trihydroyurs-12-en-28-oic acid
3{3-(trans-p-coumaroyloxy)-2a.23-dihydroxy
-urs-12-en-28-oic acid

Callus

Whole plant

Fruit gall

Regenerated'

Natural'

+
+
+

+
+
+

+
+
+

+
+

+
+
+

+
+
+

+
+

+
+
+
+
+

+
+
+

, Triterpenoids in these specimens were detected by TLC.

Since A. polygama produces interesting secondary compounds which


are specifically attractive to Felidae species, especially for cats, the callus
culture was analyzed by GC-MS, but proved, however, to produce only
a low amount of attractant for cats. On the other hand, the callus culture
usually formed various kinds of triterpenoids. The oxidation process of
these triterpenes suggested that the hydroxylation ability of the C-4 dimethyl
group in callus culture favored beta, while the natural plants, fruit galls
and regenerated plantlets had nonspecific hydroxylation ability against the
C-4 methyl group. Therefore, this ability may be useful in selective
biotransformation of cyclohexane ring triterpenoids possessing a C-4 dimethyl
group.

References
Bhandari H, Garg HS, Agrawal PK, Bhakuni DS (1990) Ursane triterpenoids from Nepeta
eriostachia. Phytochemistry 29:3956-3958
Cai OG, Oian YO, Ke SO, He ZC (1993) Regeneration of plants from protoplasts of kiwifruit
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Cheung H, Tokes L (1968) Oxygenated derivatives of asiatic acid from Dryobalanops aromatica.
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Gamborg OL, Miller RA, Ojima K (1968) Nutrient requirements of suspension cultures of
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Hazama N (1942) Felidae species and Actinidia polygama. Shizen 6:55-59
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Y. Shoyama et al.

12

Hyeon SB, Isoe S, Sakan T (1968) The structure of neomatatabiol, the potent attractant for
Chrysopa from Actinidia polygama. Tetrahedron Lett 5325-5326
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Kapoor VK, Chawla AS (1986) Biological significance of triterpenoids. J Sci Ind Res 92:503511
Kikuchi T, Matsuda S, Kadota S, Sakai Y, Namba T, Watanabe K, Dissanayake DMRB (1984)
Studies on the constituents of medicinal and related plants in Sri Lanka. I. New triterpenes
from Hedyotis awsoniae. Chern Pharm Bull 32:3906-3911
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(Choripetalae), Hoikusha, Osaka, Japan, 201 pp
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Kojima H, Tominaga H, Sato S, Ogura H (1987) Pentacyciic triterpenoids from Prunella vulgaris.
Phytochemistry 26:1107-1111
Konoshima T, Takasaki M, Kozuka M, Tokuda H (1987) Studies on inhibitors of skin tumor
promotion. I. Inhibitory effects of triterpenes from Euptelea polyandra on Epstein-Barr virus
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Konoshima T, Takasaki M, Tatsumoto T, Kozuka M, Kasai R, Tanaka 0, Rui-Lin Nie, Tokuda H,
Nishino H, Iwashima A (1994) Inhibitory effects of cucurbitane triterpenoids on EpsteinBarr virus activation and two stage carcinogenesis of skin tumor. Bioi Pharm Bull 17:668671

Linsmaier EM, Skoog F (1965) Organic growth factor requirements of tobacco tissue cultures.
Physiol Plant 18:100-127
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Biotechnology in agriculture and forestry, vol 32. Cryopreservation of plant germplasm I.
Springer, Berlin Heidelberg New York, pp 321-331
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Plant protoplasts and genetic engineering V. Springer, Berlin Heidelberg New York, pp 193214
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Actinidia polygama (Japanese name Matatabi)

13

Sashida Y, Ogawa K, Yamanouchi T, Tanaka H, Shoyama Y, Nishioka I (1994) Triterpenoids


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II Alkanna tinctoria T. (Alkanets): In Vitro


Culture and the Production of Alkannin and Other
Secondary Metabolites
C. GERARDI\ G. MITA\ E. GRILL02, G. GIOVINAZZO l , A. MICEU 2,
and P. DE LE02

1 Introduction
1.1 The Plant

The genus Alkanna (family Boraginaceae) consists of 25 species widely distributed in the Mediterranean regions and Asia. The species Alkanna tinctoria
(L.) Taush (2n = 30) also has a wide geographical distribution; in particular, it
grows wild in arid maritime areas of southern Europe. The plants are perennial herbs with prostrate bushy stems, blooming between March and May with
small (6-7mm) blue flowers (Fig. 1). The propagation of the plant occurs
naturally from seeds that are included in monospermic achenes. The percentage of seed germination is very low, which is true also for other Boraginaceae
species (Qi et al. 1993). Alkanna tinctoria has been known since ancient times
for the presence in its root of the red pigment alkannin, used since antiquity
for its therapeutical properties and as a natural dye.
1.2 Alkannin and Other Secondary Metabolites in Intact Plants

In intact plants, most alkannin derivatives are alkannin esters. The principal
alkannin esters identified are /3,/3-dimethylacryl ester and /3-acetoxy isovaleric
ester (Papageorgiou et al. 1979). The healing effect of alkannin esters has been
demonstrated on patients suffering from ulcus cruris, where after 5-6 weeks of
treatment, complete healing was observed (Papageorgiou 1978). Furthermore,
the antimicrobial activity against S. aureus and S. epidermidis was also demonstrated (Papageorgiou et al. 1979).
Alkannin has a naphtoquinonic structure and is the enantiomeric form of
another well-known important industrial pigment, shikonin, occurring naturally in the root of Lithospermum erytrhrorizon (Tabata and Fujita. 1985;
Fujita 1988; Fig. 2). A comparative analysis of the therapeutical properties of
both alkannin and shikonin performed by Tanaka et al. (1986) showed that the
antiinflammatory activities of the two compounds are identical.

[ Istituto di Rieerea sulle Bioteenologie Agroalimentari, CNR, Via Monteroni, 73100 Leece, Italy
Dipartimento di Fisiologia Vegetale, Universita di Leece, Via Monteroni, 73100 Leece, Italy

Biotechnology in Agriculture and Forestry, Vol. 41


Medicinal and Aromatic Plants X (ed. by y.P.S. Bajaj)
Springer-Verlag Berlin Heidelberg 1998

Alkanna tinctoria T. (Alkanets)

15

Fig. lA, 8. Alkanna tinctoria (L.) Tausch plant in bloom (A) and flowers (8)

Besides therapeutical uses, alkannin and other napthoquinones can be


used as natural dyes in the textile and cosmetic industries. These molecules are
being revived nowadays with renewed attention because of the increasing
interest of the market in natural products.

C. Gerardi et al.

16

Fig. 2. Enantiomeric structures of the two


naphtoquinones alkannin and shikonin present
in Boraginaceae plants. (De Leo et al. 1996)

ALKANNIN

W
0

OH

,;P'
~

OH

,'pH

c' -CH2 -CH

/ CH,

=C"

CH,

SHIKONIN
Table 1. Alkannin derivatives (mg/g dry weight) extracted from
stem and root tissues. (De Leo et al. 1992)
Plant tissue
Proximal stem
Middle stem
Distal stem
Root-suberized periderm
Secondary root

Alkannin

o
o

8.73 1.04

21.74 2.20
0.30 0.09

In a previous study, we (De Leo et al. 1992) analyzed the localization and
temporal accumulation of the pigment in plant tissues. The results revealed
that the highest accumulation of alkannin occurs during seed ripening, in
the periderm of pluriennal roots, and in the distal part of suberized stems
(Table 1).
Alkannin derivatives can be obtained by homogenization of root tissues in
chloroform. Crude extracts are purified on a Sigel column, using chloroform as
eluant, before preparative thin layer chromatography (TLC) (De Leo et al.
1992). When compared with pure alkannin, the majority of the pigments
showed a different R[, but after hydrolysis with KOH, a large fraction of the
pigments revealed the same R[ as pure alkannin. This is due to the fact that, as
has already been demonstrated (Papageorgiou and Digenis 1980), the pigments extracted from the roots contain esterified forms of alkannin. We performed gas chromatography-mass spectrometry (GC-MS) analyses of the
partially purified fractions, after KOH hydrolysis and derivatization of free
acids. The results revealed that our alkannin fractions were constituted by the
alkannin esters shown in Fig. 3.

17

Alkanna tinctoria T. (Alkanets)

COMPOUNDS

STRUCTURE

DIHYDROXYNAPHTHOQUINONIC
COMPOUNDS
(ALKANNIN AND
ITS ESTERS)

ALKANNIN

100

R=OH

N.D.

ACETYL-ALKANNIN

58.3 0.6

2-METHYL-BVTANOYLALKANNIN
ISOVALERYL-ALKANNIN

7.3 0.8

R = O-C-CH 2CH(CH,h
~

..

(Z)-2-METHYL2-BUTENOYL-ALKANNIN
(ANGELIL-ALKANNIN)

IS.9 0.1

18.6 02

Fig.3. Structure and quantitative composition (% mol) of alkannin derivatives present in purified
alkannin fraction of intact roots. (De Leo et al. 1996)

Due to the difficulties in plant cultivation and the harvesting costs, the
production of the pigment from intact roots cannot be considered a convenient
approach. Furthermore, the massive utilization of natural sources could result
in the exhaustion of wild plants, as has already happened for Lithospermum
erytrhorizon growing wild in Japan, and now almost extinct (Fujita 1988).

2 In Vitro Approaches
In vitro cultures of Alkanna tinctoria were set up to study:
1. in vitro production of regenerated plants,
2. production of alkannin from suspension cells and root cultures.

18

C. Gerardi et al.

This chapter is primarily based on our published work (De Leo et al. 1992,
1996; Mita et al. 1994).
2.1 Establishment of Cultures

2.1.1 Callus Cultures

Best results were obtained from shoot explants incubated on MS medium


(Murashige and Skoog 1962) containing 0.9-2.26.uM 2,4 dichlorophenoxyacetic acid (2,4-D). Good results were also obtained utilizing immature seeds incubated in MS medium, supplemented with 1.25.uM 6benzylaminopurine (6BAP) and 0.54.uM naphthaleneacetic acid (NAA).
Under these experimental conditions, however, calli were unable to produce
any pigment.
Suspension cultures were induced, transferring small portions of calli
in MS liquid medium supplemented with 1.uM 2,4 D and lO.uM kinetin
(MSA).
2.1.2 Root Cultures

We obtained two different root cultures (ATS23R and ATR1) that have
different origins. The A TS23R root culture was induced by inoculating 0.5 ml
of ATS23 cell suspension (growing in MSA) in 50ml of MS medium without
growth regulators (Mita et al. 1994). After induction, this root line was
sub cultivated in RC medium (Shimomura et al. 1991). ATR1 was obtained
from a root-induced callus growing in MG5liquid medium (Tabata and Fujita
1985).
2.2 Regeneration of Plants/Micropropagation

In a first series of experiments, a total number of 22 callus lines deriving both


from stem and shoot explants were utilized. All these lines were obtained by
incubating the explants in media containing 0.9-2.26.uM 2,4-D. Calli were
transferred in MS containing different concentrations of 6BAP, or in MS
without growth regulators (MS-). Incubations were carried out at 25C (under
16h of light, 123.uMs-'m-2), and sub cultivations in the same media were
performed monthly. Under these experimental conditions, only 1 of the 22
lines tested (ATF2) was able to regenerate shoots.
The regeneration ability of calli derived from immature seeds was also
tested. Callus induction was obtained by incubating the seeds in MS media
supplemented with the different hormone concentrations reported in Table 2.
Calli obtained were subcultivated in MS-, MS2, MS3, and MS4 under both
light and dark conditions. Regeneration was obtained in MS2, MS3, and MS4
only from calli that were induced in the media lacking 2,4-D. Furthermore,

Alkanna tinctoria T. (Alkanets)

19

Table 2. Hormone composition of the different media utilized for

callus induction
Medium

6BAP CuM)

NAA CuM)

2,4-D CuM)

MSI
MS2
MS3

1.25
2.50
1.25
2.50
5.00
/
/
/

0.54
0.54
/

MS4
MS5

MS6
MS7

MS8

/
/
/

/
/

/
/
/
0.45
0.90
2.26

.-,

Fig. 4. Regeneration of E4-derived shoots

regeneration was obtained only when calli were incubated in the light. One out
of eight lines (E4) was able to produce several shoots (about 20 shootslg of
callus) in MS4 medium (Fig. 4). Continuous subcultivations in MS4 medium
resulted in a decreased regeneration ability (to 0.5 shootslg of callus). Recovery of regeneration ability was obtained by alternate sub cultivations of E4 calli
in MS4 and MS- media. Shoots were isolated to induce rooting. Several media
were tested to consider also the role of IAA, NAA, indole-3-butyric acid
(IBA), gibberellic acid (GA 3), and charcoal at different concentrations. Best
results (56% rooting) were obtained for E4 shoots incubated inMS- medium
(Fig. 5); ATF2 shoots were able to produce roots (15 to 40% of rooting) in
modified MS medium containing half-strength macronutrients, 15 gil charcoal
and 2.46 or 4.90.uM IBA. In the same medium containing IAA in place of
IBA, rooting was reduced to 20%.

20

C. Gerardi et al.

Fig. 5. Rooting of E4 shoots in different media

2.3 Alkannin Production

Most of the induced suspension cultures, as well as root cultures, are able to
grow very well in the growth media but are unable to produce alkannin. The
observation that actively proliferating cells are not able to produce the desired
secondary metabolites has already been reported in other species, whereas it
has been reported that stress factors (both nutrient and enviromental stresses)
induce or enhance plant secondary metabolism (Mizukami et al. 1978; Parr
1989). We tested different parameters, in order to identify the experimental
conditions useful for alkannin production in suspension cultures.
2.3.1 Effect of Growth Regulators

The presence of 2,4-D in the culture medium strongly inhibits alkannin


production, whereas when 2,4-D was replaced by an equimolar amount of
indole-3-acetic acid (IAA) the cells started to produce alkannin derivatives
(Table 3). The pigment extracted from suspension cultures subjected to TLC
analyses after KOH hydrolysis showed the same R f as standard alkannin (Fig.
6A) and the spectrum of the pigments (in particular that obtained from E4 cell
lines) revealed the three absorption peaks that are typical of alkannin (Fig.
6B).

A/kanna tinctoria T. (Alkanets)

21

Table 3. Pigment production (;1g/g fresh weight) obtained from ATS23 and ATS23A cell lines in
MSA or MSB medium. (Mit a et al. 1994)
Cell line

Preculture
medium

Culture
medium

Alkannin
derivatives

MSA

MSA

0.64

MSA
MSA

MSB
MSA

24.08

MSA

MSB

108.20

ATS23
ATS23A
The values refer to the alkannin derivatives obtained after 15 days of preculture, followed by
further 20 days of incubation in culture medium.
MSB medium has the same composition as MSA but contains 1 JiM IAA instead of 2,4-D.

, :F/i.~~J~<.:\ . . . .. . . . .. ..

u .4000 .-----r-----.-----~----.------r-----r----~----~----~----~

[A b s 1

:
\
........................ ,........................... ,............................\ ......... ....... ..... .....
~
.
'\
,

~ '.

... ............. ......... .

" . uuuu ~~~______~__________~~~~~~~~'_'-__-~--~.~.~__~______~~~


400 . 0

Yava l ano th (n .l

700. 0

Fig. 6. A TLC of KOHhydrolyzed pigments obtained from: intact roots (lane 1); cells (lane 2);
medium (lane 3). Lane 4 is pure alkannin (Roth Gmbh, Karlsruhe, Germany). B Spectrum of
alkannin derivatives obtained from E4 cell line. (Mita et al. 1994)

22

C. Gerardi et al.

Table 4. Role of agar on cell growth and production of alkannin derivatives (pg/g fresh weight) in
Alkanna tinctoria suspension cultures
Agar(%)

Cell growth'

Alkannin derivatives

0
0.01
0.05
0.10
0.20

9.34
8.10
11.36
10.44
12.10

38.05
25.62
18.62
25.43
15.46

, The values refer to the increase in fresh weight (g) of 2.5 g starting material after 20 days in
culture.
Table 5. Role of ammonium salt on cell growth and the production of alkannin derivatives (pg/g
fresh weight) in Alkanna tinctoria suspension cultures

1
5
10
20

Cell growth'

Alkannin derivatives

6.40
10.53
10.58
8.10
7.92

132.65
13.21
21.18
23.49
25.78

, The values refer to the increase in fresh weight (g) of 2.5 g starting material.

2.3.2 Role of Nutrients

Alkannin production was stimulated when the cells were incubated in media
relatively poor in nutrients. However, in these media, cell growth is limited, so
that a convenient method is to utilize a two-stage culture system, in which two
different media are used; in the first medium (culture medium), cell growth is
stimulated, while the second medium (induction medium) promotes alkannin
production.
The presence of agar in the culture medium has been reported to be of
some importance for shikonin production in cell cultures of Lithospermum
erythrorizon (Fukui et al. 1983). We observed that the presence of agar in the
medium at a concentration of 0.2% stimulated cell growth, but had a negative
influence on alkannin production. The same effect (although more dramatic)
was observed when ammonium salts were present in the medium: in the
presence of ammonium at concentrations as low as 1 mM, the production of
alkannin was reduced by about 90% while cell growth was clearly stimulated
(Tables 4, 5). These results confirm those obtained for shikonin production
(Tabata and Fujita 1985).
2.3.3 Cell Selection

The production of alkannin varies during the culture period. In fact, in


the induction medium, suspension cultures produce alkannin at their maximum rate about 8 days after subcultivation (Fig. 7), although the pigment is
produced until about the 20th day of subculture. However, after several

Alkanna tinctoria T. (Alkanets)

23

600
500
400
Alkannin
derivatives

300
200
100

o
o

10

15

20

25

Days

Fig. 7. Production of alkannin derivatives (j.1g/g fresh weight) from ATS23A cell line after different times of incubation in induction medium (Rel)

subcultivations, alkannin production declined and sometimes stopped


completely. Although this phenomenon has not been clarified yet, we
think that it could be due to the fact that our cultures contained different cell
types with different abilities to produce alkannin derivatives. Furthermore,
successive subcultivations might operate a selective pressure towards those
cells that are able to grow better, but are unable to produce the pigment. This
aspect represents the main problem for the production of secondary
metabolites in suspension cultures. Therefore, it is very important to isolate
cell lines by repeated clonal selection or from protoplasts capable of producing continuously high quantities of pigment. We isolated one such cell
line by continuous selection of small callus portions containing red spots,
corresponding to cells able to produce alkannin derivatives (Fig. 8). This cell
line (E4) is able to grow very well in liquid medium and produces a good
quantity of alkannin (about 4-Smg/g cells) when incubated in the induction
medium.
Protoplasts were obtained from ATS23 suspension cell line subjected to
two different enzymatic treatments. Best results were obtained when cells
collected 6 days after sub cultivation were incubated in an enzymatic solution
containing 13.6mM CaCI 2, 1 % Cellulase Onozuka RS, O.S% Macerozyme RIO
and O.S M mannitol, in half-strength MS medium. The yield obtained was 2.1 X
106 protoplasts/g cells. Protoplasts were plated at a density of 1.1 X 105
protoplasts/ml in MS medium containing O.SM mannitol, 20% conditioned
medium, and 0.6% agar, utilizing the droplet system. The droplets were initially surrounded by the same liquid medium, renewed weekly with fresh
medium in which mannitol was gradually replaced by glucose and sucrose.
After about 1 month of culture, microcalli were visible, the viability of

24

C. Gerardi et a1.

,
Fig. 8. E4 callus accumulating alkannin derivatives

Table 6. Alkannin derivatives (pg/g fresh weight) obtained from


both root tissues and culture medium of A TS23R root culture.
(Mita et a1. 1994)

Medium

Root

Culture medium

RCI
RC2

289.73

61.54
30.19

196.17

The values represent the alkannin derivatives extracted from root


cultures and culture medium after 25 days of incubation in RCI or
RC2 medium.

protoplasts (monitored by Evans Blue staining) was found to be 7.4%, and


colony formation was 0.1 % (Fig. 9A-D).
2.3.4 Alkannin Production from Root Cultures

The two different lines of root cultures obtained showed different responses:
ATS23R was able to grow and produce alkannin derivatives in RC1 or
RC2 medium (Mit a et al. 1994), whereas ATR1 needs a two-stage culture
system, consisting of a culture medium (MG-5) and an induction medium
(RC1) (Table 6). In fact, ATR1 roots, when incubated in RC1 medium, were
able to produce alkannin derivatives, but their growth was completely
blocked. Interesting results were obtained with ATS23R root culture; in fact,
this line was able to release a good amount of pigments in the culture medium

Alkanna tinctoria T. (Alkanets)

25

Fig. 9A-D. Alkanna tinctoria protoplast-derived clones. A Cultured cells of A. tinctoria. B Isolated protoplasts. C Clusters derived from protoplasts after 3 weeks of culture. D Callus colonies
visible in the Petri dish after 4 weeks of culture

(Table 7). Furthermore, TLC analysis revealed no differences between the


pigments extracted from intact growing roots and those obtained from
ATS23R roots.

3 Conclusion
Alkanna tinctoria seems to be very promising both for alkannin production
from suspension cell culture and for in vitro regeneration of whole plants. The
production of alkannin from suspension cultures can hopefully be considered
for potential industrial applications. For this purpose, however, it is important

C. Gerardi et al.

26
Table 7. Pigment production and root growth in A TRI line

Culture
medium

Induction
medium

Root growth'

Alkannin
derivatives

MG5
MGS

MG5
RCI

7.3
0.1

0.0
149.5

The values represent the alkannin derivatives extracted from root


cultures after 30 days of incubation in different culture media.
, The values refer to the increase in fresh weight (g) of 2.5 g
starting material after 20 days in culture.

to develop reproducible experimental conditions and to select clones able to


produce alkannin at a high rate.
After repeated clonal selection, we isolated a cell line (E4) that was able
to produce 5 mg/g fresh weight of alkannin derivatives. This is an exciting
result, when considering that the intact roots of the plants contain 0.7 mg/g
fresh weight of pigments. Furthermore, the possibility of selecting more productive clones and of finding better experimental conditions could permit a
constant and increased production of alkannin. The regeneration of whole
plants from high productive clones could be used as a tool to create genetic
variability and to produce plants with improved production and accumulation
of alkannin in their roots.
The properties of alkannin as a dye suggest its potential industrial applications in the textile and cosmetic industries.
Acknowledgments. The authors wish to thank Dr. Carla Perrotta for critical reading of the
manuscript and helpful suggestions.

References
De Leo P, Miceli A, Gerardi C (1992) Extraction and purification of alkannin from Alkanna
tinctoria Tausch: its location in the plant and time course accumulation in the tissues. Agric
Med 122:334-339
De Leo P, Miceli A, Ronzini L, Sgarra R (1996) Chemical characterization of alkannin esterified
derivatives. AIHTEI 7:23-25
Fujita Y (1988) Shikonin: production by plant (Lithospermum erythrorhizon) cell cultures. In:
Bajaj YPS (ed) Biotechnology in agriculture and forestry, vol4. Medicinal and aromatic plants
4. Springer, Berlin Heidelberg New York, pp 225-236
Fukui H, Yoshikawa N, Tabata M (1983) Induction of shikonin formation by agar in
Lithospermum erythrorhizon cell suspension cultures. Phytochemistry 22:2451-2453
Mita G, Gerardi C, Miceli A, Bollini R, De Leo P (1994) Pigment production from in vitro
cultures of Alkanna tinctoria Tausch. Plant Cell Rep 13:406-410
Mizukami H, Konoshima M, Tabata M (1978) Variation in pigment production in Lithospermum
erythrorhizon callus cultures. Phytochmistry 17:95-97
Murashige T, Skoog F (1962) A revised medium for rapid growth and bio-assays with tobacco
tissue cultures. Physiol Plant 15:473-497
Papageorgiou VP (1978) Wound healing properties of naphthaquinone pigments from Alkanna
tinctoria. Experientia 34:1499-1501

Alkanna tinetoria T. (Alkanets)

27

Papageorgiou VP, Digenis GA (1980) Isolation of two new alkannin esters from Alkanna
tinetoria. Planta Med 39:81-84
Papageorgiou VP, Winkler A, Sagredos AN, Digenis GA (1979) Studies on the relationship of
structure to antimicrobial properties of naphthaquinones and other constituents of Alkanna
tinetoria. Planta Med 35:56-60
Parr AJ (1989) The production of secondary metabolites by plant cell cultures. J Biotechnoll0:l26
Qi MQ, Upadhyaya MK, Furness NH, Ellis BE (1993) Mechanism of seed dormancy in
Cynoglossum olfieinale L. J. Plant Physiol 142:325-330
Shimomura K, Sudo H, Sag H, Kamada H (1991) Shikonin production and secretion by hairy root
cultures of Lithospermum erythrorhizon. Plant Cell Rep 10:282-285
Tabata M, Fujita Y (1985) Production of shikonin by plant cell cultures. In: Day P, Zaitlin M,
Hollander A (eds) Biotechnology in plant science. Academic Press, New York, pp 207-218
Tanaka S, Tajama M, Tsukada M, Tabata M (1986) A comparative study on anti-inflammatory
activities of the enantiomers, shikonin and alkannin. J Nat Prod 49:466-469

III Amebia euchroma: In Vitro Culture and


the Production of Shikonin and Other
Secondary Metabolites
O.V.

ZAKHLENJUK

and V.A.

KUNAKH

1 Introduction
1.1 Distribution and Importance of the Plant

The genus Arnebia (family Boraginaceae) comprises around 25 species. Plants


of Arnebia euchroma (Royle) J onst. are perennial grasses of the Alpine belt,
distributed in the Pamirs, Tien Shan, western Tibet, and in the Himalayas (Fig.
1; Chukavina 1984).
In some reports Arnebia euchroma (Royle) J onst. is mentioned as
Macrotomia euchroma (Royle) Pauls. because of the fact that some taxonomic
literature gives these two botanical names as synonyms for the same species,
but unfortunately attributes the latter to different genera (Cherepanov 1981;
Chukavina 1984). According to Cherepanov (1981), this species should be
attributed to the genus Arnebia.
Plants of the genus Arnebia , as well as some other Boraginaceae species
from the genera Lithospermum, Onosma, Echium, and others, provide
the source of the naphthoquinonous red pigment, shikonin. Shikonin has
been known since ancient times as a dye used for silks and food products. At
the same time, shikonin is recognized as a remedy, showing a wide range
of effects. It possesses antibacterial and antifungal activities, and exhibits
antiinflammatory and wound-healing properties. The antiallergic, antipyretic,
antihydropic effects of shikonin and its derivatives, as well as the antineoplastic activities of shikonin have also been demonstrated (see Terada et al.
1990).
Wild plant species from the Boraginaceae family, accumulating shikonin,
fail to provide a sufficient raw-material base for commercial production, for
which reason tissue culture was derived from several species of this family.
However, the commercial production of shikonin is restricted so far to
Lithospermum erythrorhizon cells cultured in Japan (Fujita 1988). Shikonin
produced in this way by the Mitsui Petrochemical Industries since 1984 is
widely used in the cosmetic industry.

Institute of Molecular Biology and Genetics, National Academy of Sciences of Ukraine, Kiev
252143, Ukraine
Biotechnology in Agriculture and Foresty, Vol. 41
Medicinal and Aromatic Plants X (ed. by y.P.S. Bajaj)
Springer-Verlag Berlin Heidelberg 1998

Arnebia euchroma

29

Fig. 1. Arnebia euchroma (Royle) Jonst. 1 General appearance of the plant; 2 lower part of the petal; 3 fruit
nutlet

1.2 Naphthoquinone Accumulation in the Plants

Pimenova and Tareeva (1980) investigated various A.euchroma plant


populations collected from 27 regions of Tien Shan and the Pamirs in
Tajikistan. Shikonin content in root dry biomass varied from 0.39 to 1.60% in
the Tien Shan region and from 0.43 to 2.47% in the Pamirs region. Pigment
content was not lower than 1 % in some populations from Alpine areas of the
Pamirs. Nevertheless, there was no precise correlation between pigment content and geographical origin of the plants. At the same time, the shikonin
content depended on the age of the plants within the same population. Middleaged plants with 6-10 flowering stems showed the highest possible shikonin
level.
The naphthoquinone fraction isolated from intact plant roots
of A.euchroma was investigated. Davydenkov et al. (1991) and
Zakhlenjuk et al. (1993) reported the shikonin derivatives isobutylshikonin,
dimethylacrylshikonin, acetylshikonin, deoxyshikonin, and hydroxyisovalerylshikonin (Fig. 2). The major pigments are isobutylshikonin and dimethylacrylshikonin (Fig. 3a; Davydenkov et al. 1991). Shikonin is present as
traces.

o.v. Zakhlenjuk and v.A. Kunakh

30
Naphthoquinones:

~
YV~
OH

DS

(R=H)

(R=OH)

AS

(R=OCOCH3)

IBS

(R=OCOCH(CH312)

DMAS

(R=OCOCH=C(CH312)

HIVS

(R=OCOCHzC(OH)(CH312)

Benzoquinones:
R

Echinofuran B

(R=H)

Echinofuran C

(R=OCOCH=C(CH312)

Fig. 2. Secondary metabolites identified in A.euchroma plant (naphthoquinones) and


suspension tissue culture (naphthoquinones and benzoquinines). DS Deoxyshikonin; S
shikonin; AS acetylshikonin; IBS isobutylshikonin; DMAS ,8,,B-dimethylacrylshikonin; HIVS ,8hydroxyvalerylshikonin

2 In Vitro Culture Studies


2.1 Establishment of Tissue Culture

A.euchroma tissue culture was pioneered in Russia by Rabinovich and


Davydenkov in 1987 (Davydenkov et al. 1991). To initiate callus tissue they
used dormant bud meristem of plants collected in Tajikistan (the western
Pamirs). Callus tissue was generated on agarized medium, containing macroand micronutrients, thiamine, pyridoxine, nicotinic acid, and Ca-pantothenate
according to Murashige and Skoog (1962), supplemented with kinetin and
2,4-D (1 mg/l of each) and casein hydrolysate (100mg/l). After 2 months, the
calli were transferred into liquid medium with the same composition for
several passages, every 30 days, followed by transfer into modified Linsmaier
and Skoog medium (Davydenkov et al. 1991) with composition as follows (in
mg/l): KN0 3 3800, MgS0 47HzO 370, CaClz'6H20 440, KH2P0 4 170,
MnS0 4-4H20 22.3, ZnSO{7H20 8.6, Na 2Mo0 42H20 0.25, CoClz6HzO 0.025,

31

Arnebia euchroma

4,5

4,5

10

Retention time, min

10

Retention time, min

Fig. 3a,b. Densitograms of shikonin and its derivatives isolated from intact root (a) and tissue
culture (b) of A.euchroma. 1 Shikonin; 2 deoxyshikonin; 3 acetylshikonin; 4 dimethylacrylshikonin; 5 isobutylshikonin. Along the horizontal line time of scanning, min

H}BO} 6.2, KI 0.S3, CuS0 4 5H20 0.3, NaEDTA2H2 0 37.3, FeS0 4 7H20 27.S,
thiamine 0.4, nicotinic acid 0.5, pyridoxine 0.5, mesoinositol SO, indole acetic
acid 0.3, kinetin 1, and sucrose 3%.
Rabinovich and Davydenkov kindly provided several laboratories
with cell lines of established suspension tissue culture of A.euchroma
(strains 75 and 80) to carry out investigations. Independently, A.euchroma
suspension tissue culture was obtained only in China (Dong et al. 1993).
Various results of in vitro studies conducted on A.euchroma are summarized
in Table 1.
2.2 Secondary Metabolites in Amebia euchroma Tissue Culture
Cells of suspension culture after 1 year of passaging built up greater amounts
of shikonin, deoxyshikonin, and acetylshikonin as compared to intact
plant, while the accumulation of dimethylacrylshikonin and isobutylshikonin progressed less extensively than in plants (Fig. 3b; Davydenkov
et al. 1991). In addition to the above-mentioned shikonin derivatives, an
insignificant amount of hydroxyisovalerylshikonin was later identified in
the A.euchroma cell line SO-2 (Zakhlenjuk et al. 1993). Besides naphthoquinones, the cultured cells accumulated yellow pigments identified as
benzoquinones (echinofuranes Band C; Fig. 2; Davydenkov et al. 1991).
Accumulation of these six naphthoquinones, as well as of two forms

32

O.v. Zakhlenjuk and v.A. Kunakh

Table 1. Summary of various in vitro studies on Arnebia euchroma

Reference

Results and remarks

Davydenkov et al. (1991)

Initiation of tissue culture. The composition of the


naphthoquinone fraction in suspension culture differed from
that in the intact plant. Accumulation of benzoquinones in
cultured cells, in contrast to the intact plant was also found
Cell aggregate cloning was effective in increasing the production
of shikonin in suspension culture. Subsequent selection for the
most colored suspension samples in every passage failed to
stabilize and increase the productivity of cell culture. Selection
of small cell aggregates during several subcultures proved
effective in enhancing shikonin production
The best of four other media tested to promote shikonin
production in suspension culture was that proposed by
Davydenkov et al. (1991). Kinetin may be excluded from the
medium
Polyvynilpirrolidone (1 gil) promoted cell growth and shikonin
accumulation in suspension culture. Na,EDTA (1-2%) and
Triton X-100 (1-5%) stimulated the accumulation of shikonin
in culture medium when added at the end of the growth cycle
for 1-3h
Increase in shikonin content in suspension culture was found to
correlate with increase in mean nucleolar size, but not mean
nucleolar number, per nucleus
Growth character and shikonin accumulation dynamics were
found to have similar patterns in cell suspension irrespective of
cultivation in flasks or bioreactor. Biomass and shikonin yields
reached correspondingly 9.45 gil (dry wt.) and 1.93 gil after 30
days of cultivation in the bioreactor
The addition of 102 ,uM ethyl jasmonate and 10 ,liM 12-oxophytodienoic acid to suspension culture for 5-6 days resulted,
respectively, in 12- and 8-fold increases in the shikonin level.
Stimulation of shikonin biosynthesis was coupled with cell
growth inhibition
Long-term stepwise selection of suspension culture to pfluorophenylalanine (PFP) (20-100mg/l) resulted in significant
inhibition of shikonin production and changes in the
naphthoquinone spectrum. The PFP-resistant cell line revealed
no significant overproduction of free phenylalanine
PFP-resistant cell line maintained in PFP-free medium for 2 years
exhibited decreased level of shikonin accumulation compared to
control cell line. Indole butyric acid stimulated shikonin
biosynthesis in a resistant line
Shikonin yield in two-step cultivation of suspension culture
reached 1 gil, while in one-step cultivation it was not larger than
140 gil. Extraction of shikonin in situ with paraffin oil and
hexadecane at the second step of cultivation did not result in
increased shikonin production
Optimized macroelement composition of the medium and some
selection procedures (mainly the transferring of tissue in the
stationary growth phase) were found to increase shikonin
production. Naphthoquinone, as well as benzoquinone spectra
in tissue culture, corresponded to that already reported.
Triterpene saponin and coumarin accumulation in tissue culture
were reported for the first time

Zakhlenjuk et al. (1991)

Shepel et al. (1991)

Zakhlenjuk et al. (1992)

Zakhlenjuk (1992)
Dong et al. (1993)

Bychkova et al. (1993)

Zakhlenjuk et al. (1993)

Zakhlenjuk (1994)

Kozlovtseva et al. (1994)

Sokha (1996)

Arnebia euchroma

33

of benzoquinones, in cell line 75 was recently confirmed by Sokha (1996). The


major red pigment in this cell line proved to be acetylshikonin. The increase in
cell line productivity was associated with an increased relative amount of
acetylshikonin, while the levels of other naphthoquinones and also
benzoquinones decreased.
The production of other secondary metabolites in A.euchroma tissue culture - triterpene saponins and coumarins - was also reported (Sokha
1996).
During the maintenance of suspension culture, some part of the
pigments accumulated in nutrient fluid. According to our results, this portion could reach 30-40% of the total amount of pigments. Microscope
analysis showed that medium precipitate contained round-shaped crystals
in different shades of red. The qualitative reaction of the isolated crystals
with a 5% solution of KOH suggests their naphthoquinonous nature.
Along with the red crystals in the precipitate, yellow comb-shaped ones
may be present, whose number tends to increase when the culture turns
brown. There are no earlier reports of naphthoquinone accumulation in a
crystallized form during culturing of other plant tissues that synthesize these
pigments.
2.3 Selection for Shikonin-Overproducing Tissue Culture Variants

2.3.1 Mass Culturing


In selecting suspension culture for higher productivity during mass culture, we

used as a criterion the general culture coloring, namely, the coloring of both
tissue and culture medium. For further subculturing, the flasks with culture
(here designated subcultures) showing the most intensive coloring were
picked out. However, the experience of several years of culturing the initial
strains 75, 80, and some variants derived from them had shown that this
method of selection, in spite of temporary positive results, finally proved
ineffective. As is seen from Fig. 4, the productivity of strain 80 remained
insignificant and clones generated from it exhibited a progressive decline in
productivity despite consistent screening of the most intensely colored samples of subcultures for transfers.
The failure of this selection technique was also confirmed by the results of
two alternative, simultaneously performed for 10 passages, selections: one for
increased, the other for reduced shikonin content. The results for first three
passages summarized in Table 2 reveal great differences in the shikonin content in "offspring" of the subculture selected for transfer in each passage.
Average shikonin content between all subcultures in each passage during
positive selection exceeded that for negative selection, but this difference
disappeared after four to six passages. Negative selection appeared to be even
more effective than positive, as it is interesting that by constant selection of
the least colored subcultures "offsprings" emerge that exceed not only the
original culture but the variants of positive selection as well in productivity.

o.v. Zakhlenjuk and v.A. Kunakh

34

50

I :::~~
_--3 I

40

~d'

30

~
'"

20

10
A

- -.- - -. -

-+- --.

10

15

L-~~~~L-~~~~L-~~~~L-~~L-~

20

Passage number
Fig. 4. Shikonin content in the course of sequential passages of A.euchroma tissue culture. 1
Clone 80-1; 2 clone 80-2; 3 original strain 80. Each point represents average of three to five

analyzed subcultures. Shikonin content was estimated in culture fluid

Table 2. Shikonin content in individual subcultures during both

positive and negative selection of A. euchroma tissue culture


Cell
line

75

Starting population of
subcultures"

127.5/'
85.0
67.5
37.0 \,
(79.2)

(+ )

(-)

80-2

(+)
260.0/'
200.0
173.0
(211.3)\,
(- )

Shikonin content (mg/I)


1st passage

2nd passage

3rd passage

111.0
109.0
103.0
47.0
(92.5)

132.5
110.2
81.2
67.0
(97.7)

90.0
85.0
52.0
45.0
(68.0)

34.0
33.5
27.0
26.0
(30.1)
217.5
170.0
165.0
(184.2)

45.2
43.7
39.5
32.5
(40.2)
186.2
175.0
131.2
(164.2)

51.0
42.0
40.0
30.0
(40.7)
255.0
190.0
175.0
(206.6)

223.7
155.0
111.9
(163.5)

138.7
131.2
103.7
(124.6)

185.0
165.0
120.0
(156.6)

" Positive (+) and negative (-) selection. The material of subcultures that are underlined was used entirely for subsequent
subculturing. Parentheses indicate mean shikonin content for all
subcultures in each passage.

Arnebia euchroma

35

Such results might reveal wide range of cell chemotype variability in the
culture.
The results indicate the necessity of searching for additional selection
criteria during mass cUlturing. It was suggested that the selection of small
uniform cell aggregates might be useful for this purpose. The approximately
equal size of cell aggregates may be a decisive factor which possibly indicates
similar or relatively more synchronized cell differentiation processes within
tissue structures. Brought together, such cell aggregates may undergo more
coordinated development, thus approximating the cell culture to the status of
intact plant tissue. This suggestion is substantiated by our results (see Sect.
2.3.3).
2.3.2 Cell Aggregate Cloning

For cloning the cell aggregates of two cultured strains, 75 and 80, we used cell
clumps of 1-2 mm in size, thus obviating the need for application of feeder
layers or conditioned media. Due to the peculiarities of the cloning technique
(aggregate maintenance in drops of nutrient medium supplemented with mineral oil), the variability of aggregates in pigment accumulation manifests itself
already at the first step of cloning, thus making possible early rejection of
undesirable variants which make the oil yellow or orange, or fail to color it at
all. The latter account for 25%. The aggregates which color oil light pink make
up about 50% with the remaining ones deep pink and red. More productive
clones were obtained only from the strain 80, which, in contrast to strain 75,
was characterized by greater tissue density and less vigorous growth, as well
as by the availability of a large number of small, dense globular aggregates
that were used for cloning. Cell aggregates of strain 75 proved friable and
predominantly of lamella structure. The results indicate that dense, small
cell aggregates are more effective for cloning. It is also obvious that the
cloning procedure may be effective if the initial size of cell aggregates reaches
1-2mm.
Subsequent long-term selection of the most intensively colored subcultures of new clones failed to promote stability in increased productivity level
(Fig. 4).
2.3.3 Selection of Different Fractions of Tissue Culture

To support the productivity of clone 80-2 suspension culture we used a


method technically more simple than cloning - the periodically repeated selection of small cell aggregates followed by their joint subsequent culturing.
Uniform cell aggregates (colored, 1-2mm in size) were picked out from various
subcultures and maintained in the same way as the stock culture. This selection
was carried out in 14 passages within a year with various intervals at
which selection of the most intensively colored subcultures was carried out.
Despite the fact that average subculture productivity continued to vary from

36

O.v. Zakhlenjuk and V.A. Kunakh


30r-----------------------------------~

25

20

10

20

40

60

80

100

120

140

160

180

Shikonin. mg / L

Fig.5. Distribution of individual subcultures in terms of shikonin content in A.euchroma cell line
80-2 during long-term selection by most intensive coloring. N N umber of subcultures; V index of
variation; M mean value of shikonin content (57 mg/I)

passage to passage, its level gradually increased. As compared with the


period when subculture selection was restricted to culture coloring (Fig. 5), the
distribution of shikonin content when two types of selection were combined
(Fig. 6) was characterized by relatively reduced variability, and practically
corresponded to the normal one. Shikonin content ranged from 180 to
559mg/l.
After the periodic selection of a small cell aggregate fraction was
finished, but subculture selection for the most intensive coloring was continued, within a year productivity decreased somewhat. Shikonin content, however, varied within the range of 150 to 350mg/l, i.e., did not drop to the initial
level (see Fig. 5). Increase in productivity may result from replenishing the
culture with productive cell chemotypes by repeated selection of small uniform cell aggregates, as well as due to the appearance of stable modifications
in control mechanisms of pigment biosynthesis in such a cell aggregate
population.
During suspension culture growth, a substantial amount of biomass
adheres to the walls of the culture vessel. Analysis revealed that this fraction
of strain 75 may accumulate up to 70% of the pigments. Detachment of
such a fraction resulted in more productive variants of strains 75 (Fig. 7a) and
80-2 (Fig. 7b). However, further subculture selection for the most intensive

37

Arnebia euchroma
35

30

N=37
V=13%

25

eI:

'"

20

~
;:;

15

B
:;
en

10

0
100

200

300

400

500

600

Shikonin, mgIL
Fig. 6. Distribution of individual sub-:ultures in terms of shikonin content in A.euchroma cell line
80-2 during alternation of selection by the most intensive coloring with selection of fraction of
small colored cell aggregates. N Number of subcultures; V index of variation; M mean value of
shikonin content (349 mg/I)

coloring resulted in a progressive reversal of productivity to the initial


level. When the selections of such a fraction were repeated the pattern was the
same.
The biomass adhering to the glass represents a mixture of fibrils, large
old cells, and small cells and cell aggregates. The latter two appear to give
rise to novel cell lines. Thus, the same mechanism might underlie the positive
effects of selection of adhered biomass as well as of small submerged cell
aggregates.
2.3.4 Color Tint as a Selection Criterion
A.euchroma suspension culture tends to change color during passaging. A shift
from red to yellow-brown was observed to be coupled with reduced shikonin
content. This fact predetermined the choice of a pink-lilac tint in
the subculture as a selection criterion. This selection procedure was also
associated with an increase in inoculum size (from 1.2-1.8g/1 dry wt. to 1.82.8g/1 dry wt.). Shikonin content of lS0-3S0mg/1 before the start of the experiment increased greatly after such selection and was maintained at

O.v. Zakhlenjuk and v.A. Kunakh

38
700
600

='S

]
:E
CI)

500
400
300
200
100
0
450
400
350

0:2OIl
8

300

='S

250

200

CI)

150
100
50

10

15

20

25

Passage number
Fig.7a,b. Shikonin content in A.euchroma tissue culture. a Strain 75. b Cell line 80-2. 1 Control;
2 cell lines resulting from the selection of cells and cell aggregates adhering to the flask walls. Each
point represents average of three replicates

the increased level for a year (Table 3). Microscopic monitoring failed to
reveal yellow crystals in the culture fluid. Currently, productivity in the best
subcultures may reach 1 gil for 14 days or 71.4mg/l per day. This result is
comparable to that reported for another A.euchroma suspension culture
(Dong et al. 1993). Authors claimed that the shikonin yield for 30 days of
culture growth in bioreactor was estimated to be 1.93 gil, which is equal to
64.3 mgll per day.
Thus, selection by color tint proved more effective than selection
only by the most intensive general coloring. Such selection seems to be
more useful in rejecting those subculture samples where biosynthesis of the
yellow pigments or other metabolites competing with naphthoquinones IS
enhanced.

Arnebia euchroma

39

Table 3. Shikonin content in A. euchroma tissue culture during selection of subcultures by tint of
coloring
1995

(year
quarter)
I
II
III

IV

Shikonin
content
(mg/I)"
390.8
672.5
940.0
688.8

56.2
40.0
36.0
44.3

Range
of variationb

Shikonin
content per
dry weight (%)

Biomass
dry weight
yield (g/I)

295.8
525.0
904.0
568.0

2.9
5.5
7.7
6.3

13.5
12.2
12.2
11.1

468.0
702.0
976.0
813.7

" Mean value for the quarter year.


b Variation in individual subcultures by shikonin content.

2.3.5 Selection for Resistance to p-Fluorophenylalanine

Proceeding from the assumption that phenylalanine may be one of the


precursors of naphthoquinone biosynthesis, we obtained the variant of
clone 80-2 of A.euchroma tissue culture resistant to p-fluorophenylalanine (PFP) using long-term stepwise selection (20 mg/I-100 mg/l
PFP) (Zakhlenjuk et al. 1993). The resistant variant, however, was distinguished by significantly decreased productivity, both with and without
PFP in the medium. Free phenylalanine content in the PFP-resistant line increased only by 1.1-1.8 times, while a four-fold increase in lysine content
was observed at the same time. The PFP-resistant line was also distinguished
by changes in the naphthoquinone spectrum. As compared with the
control line, where significant amounts of isobutylshikonin, dime thylacrylshikonin, and acetyshikonin, as well as insignificant amounts of
deoxyshikonin, hydroxyisovalerylshikonin, and shikonin were accumulated,
the PFP-resistant cell line was recorded as building up an insignificant
amount of acetylshikonin and traces of deoxyshikonin and shikonin. PFPresistance was simultaneously coupled with enhanced biosynthesis of
benzoquinones.
PFP used at a high dose (lOOmg/l) during three successive passages was
also reported to decrease shikonin accumulation in A.euchroma tissue culture
(strain 75) (Sokha 1996). However, the shikonin content in this cell line was
found to increase by 14 % after the stress pressure was removed. According to
our results, the PFP resistant cell line maintained without PFP in medium for
2 years showed only partly restored productivity (25% of control level). At the
same time, the substitution of indole acetic acid (IAA) for indole butyric acid
(IBA) in the medium, with simultaneous removal of kinetin, considerably
stimulated shikonin biosynthesis in the PFP-resistant variant, while the control
line response under the same conditions was weak (Fig. 8). The resistant
variant transferred from the medium supplemented with IBA back into the
control medium with or without PFP had decreased shikonin level, typical for
this variant (Zakhlenjuk 1994). Drastic and reversible changes in shikonin
biosynthesis upon simple hormonal modifications in the nutrient medium are
inconsistent with the idea of the mutational nature of metabolic shifts in PFPresistant cells.

40

O.Y. Zakhlenjuk and Y.A. Kunakh


120

"0
l:l

c:

120

100

100

80

80

60

60

40

40

20

20

""'0
<i<

.5c:
0

~
CIl

Fig. Sa,b. Shikonin content in A.euchroma cell line 80-2 (a) and its PFP-resistant variant (b). J
Control medium; 2,4 medium containing indole butyric acid (1 mg/l); 3 control medium, containing PFP (lOOmg/l)

2.4 Effect of Environmental Factors

2.4.1 Growth Regulators


In an experiment with cell line 80-2, it was shown that substitution of kinetin
by 6-benzylaminopurine (BAP) at the same or at a lesser concentration in
nutrient medium encouraged increased productivity. Figure 9 represents the
results of comparative studies of the effects of various cytokinin concentrations coupled with IAA levels of 0.3 mg/l (control medium) and 1 mg/I. The
biomass increment in every case either remained at the control level or even
rose somewhat. The shikonin content essentially increased with the IAA +
BAP combinations: 0.3mg/1 IAA + 0.1mg/1 BAP and 1mg/1 IAA + 0.1mg/1
BAP. The results show the differing efficiency of cytokinins in naphthoquinone biosynthesis.
As other authors reported (Shepel et al. 1991; Koslovtseva et al. 1994), cell
strain 75 of suspension culture was able to grow in medium devoid of kinetin.
Under such conditions, its productivity reached 140mg/l, while under our
conditions (the same medium supplemented with kinetin), it hardly exceeded
60mg/I. Nevertheless, cell line 80-2, in our results, was found to reach a much
higher level of productivity after selection in the same medium containing
kinetin.
2.4.2 Macroelement Composition
Macroelement composition in the nutrient medium used (see Sect. 2.1) was
changed as follows (mg/l): KN0 3 2000, Ca(N03)2 440, NaH2P0 42H20 85,

Arnebia euchroma

41
150

150

"8

100 - ----

,--..-0.:11 .,...,--------

---

100 ]

---------

c:

''"'E""

"""o

<ff:<:

o
:.0

'2
o

....

,....

cs

50

50

0.1 1.0 2.0

0.1 1.0 2.0

KIN

BAP
(mgfL)

(mgfL)

(0.3 mgfL IAA)

0.1

1.0

KIN
(mgfL)

0.1

:.2

(Il

1.0

BAP
(mgfL)

(1.0 mgfL lAA)

Fig. 9. Effect of kinetin (KIN), 6-benzylaminopurine (BAP), and indole acetic acid (fAA) on the
increase in biomass and shikonin content in A.euchroma cell line 80-2. K Control. Shikonin
content was estimated in culture fluid

NaZS04 500, MgS0 47HzO 370. After transfer of cell line 80-2 into the medium
with this composition of macroelements, increased culture productivity was
registered. Shikonin content in the first two passages comprised 842.5 and
814.4mg/l, respectively, the control, however, as little as 688.8 and 453.1 mg/l,
respectively.
Optimized macro element composition, proposed by Sokha (1996) for
medium used to cultivate cell strain 75, was as follows (mg/l, lower level):
KN0 3 3750, Ca(N03 )z 500, KCI 175, KH zP0 4 110, KzHP0 4 70, MgS0 4 6HzO
470. This medium, supplemented with 4.5% sucrose, was reported to increase
shikonin accumulation by 50%.
2.5 Differentiation

Cell lines 80 and 80-2 of suspension culture exhibited significant potentiality


for rhizogenesis, which asserts itself after substituting IAA for NAA in the
nutrient medium, with simultaneous kinetin removal. Strain 75 failed to display such a capacity. It is noteworthy that in the presence of kinetin this auxin
replacement did not induce root formation in line 80-2 as well. The PFPresistant variant of cell line 80-2 lost the ability for root formation under

42

o.v. Zakhlenjuk

and v.A. Kunakh

similar conditions. Root formation in cell line 80-2 did not appear to correlate
with shikonin content increase (see Fig. 8).

3 Summary and Conclusion


More productive variants of Arnebia euchroma suspension culture were obtained via cloning of cell aggregates (1-2mm in size), repeated selection of
fraction of colored cell aggregates of the same size, as well as selection of the
culture fraction adhering to the walls of the culture vessel. Subsequent growth
of novel cell lines by means of permanent selection of subculture samples by
most intensive general coloring of tissue and cultural fluid failed to increase or
stabilize productivity. At the same time, selection by pink-lilac color tint
favored increase in culture productivity. Before the start of selection procedures the shikonin content ranged from 0.2 to 1.4% dry weight. As a result
mainly of periodical selection of the fraction of small colored cell aggregates
followed by selection for coloring tint, the shikonin content varied from 2.9 to
7.7% dry weight. Productivity of the best subculture samples amounts to 1 gil
for 14 days. Long-term selection by cell resistance to p-fluorophenylalanine to
increase culture productivity proved ineffective.
Considerable and easily reversible changes in the biosynthetic activity
of A.euchroma cultured cells caused by environmental agents, macroelements, and phytohormones in particular, support the idea of epigenetic level for naphthoquinone biosynthesis control. At the same time, the
relatively stable increase in productivity on long-term selection using the same
nutrient medium may indicate the appearance of steady modifications of
control mechanisms ensuring naphthoquinone biosynthesis. This may be
caused by culture enrichment with productive cell chemotypes, which
resulted from repeated selection of small uniform cell aggregates. More
synchronized processes of cell growth and differentiation in such cell
aggregates may provide a better balance in metabolic events. Cell differentiation in vitro seems to be the major reason for the persistence of
cell heterogeneity in terms of biosynthetic capacity as well as other characteristics. Therefore, to select more productive tissue cultures, selection criteria
should be sought taking into due account cell differentiation characteristics
in vitro.

4 Protocol
4.1 Maintenance of Tissue Culture
Tissue cultures of A.euchroma (strains 7S, 80, and resultant variants) were maintained in modified
Linsmaier-Skoog medium (Davydenkov et al. 1991) in 2S0-ml Erlenmeyer flasks containing SOml

43

Arnebia euchroma
of medium, with agitation on shaker (80-100 rpm), in the dark at 24
cycle was 14 days.

:!:

1C, Duration of growth

4.2 Cell Aggregate Cloning


Cell aggregates 1-2 mm in size were selected and placed individually into separate drops of
nutrient medium (20 J11) supplemented with 2.5 J1l of liquid paraffin (carbon number> 18C) and
placed onto Petri dishes. After 5 days, cell aggregates among drops distinguished by the most
intensively colored oil were transferred into plate wells containing 0.5 ml of nutrient medium and
0.2ml of oil. One week later, biomass from wells was transferred into 50-ml flasks containing 5ml
medium and 2 ml oil, and left for 14 days. Each step was run in an intermixing regime, using the
same medium.

4.3 Analysis for Shikonin Content


Aliquots of cell suspension (tissue and culture fluid were sampled in proper proportions) or
exclusively of culture fluid (resulting from filtration of culture through a nylon mesh with 100-J1m
pore size) were mixed with acidified alcohol (1 ml of concentrated H 2SO';11 EtOH). In the case of
cell suspension analysis, filtered tissue was exhaustively extracted with fresh portions of alcohol.
Pooled alcohol extracts were exposed to either liquid paraffin or hexane under vigorous agitation
for 5 min. Pigments found either in paraffin or hexane were hydrolyzed with 5% KOH solution.
Alkaline solutions were examined photocolorimetrically. As a standard the alkaline solution of an
authentic sample of shikonin was used.
Acknowledgments. The authors wish to thank S.A. Rabinovich for kindly giving strains 75 and 80
of suspension culture of Arnebia euchroma (Royle) Jonst., and for the authentic sample of
shikonin, and V.I. Adonin for technical assistance.

References
Bychkova TP, Nanenina EV, Bersin VB, Miroshnikov AI (1993) Influence of jasmonic acid and
12-oxo-phytodienoic acid on the biosynthesis of shikonin in the Arnebia euchroma cell culture.
Bioorg Chern 19:1008-1012 (in Russian)
Cherepanov SK (1981) Vascular plants in the USSR. Nauka, Leningrad (in Russian)
Chukavina AP (ed) (1984) Flora of Tajik SSR, vol 7. Nauka, Leningrad (in
Russian)
Davydenkov VN, Patudin AV, Popov YuG, Rabinovich SA, Miroshnikov AI (1991) Cell culture
of Arnebia euchroma (Royle) Jonst. - novel source of shikonin production. Him Farm Zh
1:53-55 (in Russian)
Dong J, Ye H, Wu X, Li G, Wu Z, Chen J (1993) Studies of suspension culture and fermentable
culture of Arnebia euchroma. Acta Bot Sin 35:57-61 (in Chinese)
Fujita Y (1988) Shikonin: Production by plant (Lithospermum erythrorhizon) cell cultures. In:
Bajaj YPS (ed) Biotechnology in agriculture and forestry, vol 4. Medicinal and aromatic plants
I. Springer, Berlin Heidelberg New York, pp 225-236
Kozlovtseva LV, Zaitseva GV, Strogov SE (1994) Suspension culturing of macrotomia Arnebia
euchroma cells. Optimization of conditions for shikonin production. Biotechnologia 3:24-26
(in Russian)
Murashige T, Skoog F (1962) A revised medium for rapid growth and bioassays in tobacco tissue
cultures. Physiol Plant 15:473-497

44

O.V Zakhlenjuk and VA. Kunak: Arnebia Euchroma

Pimenova ME, Tareeva NV (1980) Shikonin content variability in the underground organs of
macrotomia dyeing. Rastit Resur 16:82-86 (in Russian)
Shepel LI, Erokhina LV, Strogov SE, Zaitseva GV, Rabinovich SA, Davydenkov VN (1991)
Suspension culturing of macrotomia dyeing cells. In: Progressive industrial methods for the
medical industry. Part 2, NPO "BIOMASH", Moscow, pp 23-25 (in Russian)
Sokha VI (1996) Shikonin sources in tissue culture. PhD Thesis, Chemical Pharmaceutical
Institute of St. Petersburg (in Russian)
Terada A, Tanoue Y, Taniguchi H (1990) Chemistry of shikonin, ancient purple pigment and its
derivatives. J Syn Org Chern Jpn 48:866-875 (in Japanese)
Zakhlenjuk OV (1992) Correlation between nucleolar number, nucleolar size and productivity of
macrotomia Arnebia euchroma cultured cells. Cytologia 34:69 (in Russian)
Zakhlenjuk OV (1994) Some peculiarities of p-fiuorophenylalanin-resistant macrotomia dyeing
cell culture. Biopolymery i Kletka 10:32-37 (in Russian)
Zakhlenjuk OV, Vidmachenko TV, Kunakh VA (1991) Shikonin accumulation dynamics during
long-term selection of Arnebia euchroma suspension culture. Abstr 1st All-Uinion Symp New
Method Plant Biotech, Pushchino, pp 61-62 (in Russian)
Zakhlenjuk OV, Vidmachenko TV, Kunakh VA (1992) Effect of some abiotic elicitors on the
shikonin accumulation in macrotomia suspension culture. Abstr 3rd Ukr Confr Med Bot, Kiev,
p 62 (in Russian)
Zakhlenjuk OV, Vidmachenko TV, Kunakh VA, Rabinovich SA, Davydenkov VN (1993) Comparative characteristics of shikonin accumulating Arnebia euchroma suspension culture and its
p-fiuorophenylalanine-resistant variant. Acta Hortic 330:293-298

IV Campanula (Bellflower) Species: In Vitro


Culture, Micropropagation, and the Production of
Anthocyanins, Polyacetylenes, and Other
Secondary Metabolites
K. BRANDT1 and K. ISHIMARd

1 Introduction
1.1 Distribution and Importance of the Plant

The genus Campanula (family Campanulaceae) comprises approximately 300


species distributed across the northern hemisphere (Cook 1951), many of
these in mountainous areas. The genus generally inhabits meadow and
subalpine regions, many species requiring full sun for optimal development.
All species are herbaceous, and the name refers to the bell-shaped, blue
flowers of the majority of the species. The species are perennials, biannuals, or
annuals.
A few species, Campanula glomerata, C. persicifolia, C. rotundifolia, C.
bononiensis, C. sibirica, and C. patula have been used locally for preparation
of traditional drugs in Russian folk medicine (Barnaulov et al. 1983, 1984 and
references therein), and, in Italy, similar use has been made of C. medium, C.
cervicaria, C. rotundifolia, C. latifolia, and C. trachelium (Gastaldo 1978).
Preparations were used to treat epilepsy, nervous diseases, coughs, headache,
rheumatism, and inflammation. Many of the medicinal qualities attributed to
these species are similar to the use in oriental medicine of drugs made from the
closely related Platycodon grandifiorum (Ozaki 1995; Tada et al. 1995a), a
species originally classified in the genus Campanuia, and from species of the
allied genera Adenophora (Kuang et al. 1991) and Codonopsis (Wang et al.
1995). Commercial cultivation of Campanula is mainly for ornamental
purposes, the value of the European production being estimated in the range
of 25 million DM per year for pot plants (1. Andersen, pers. camm.) plus a
minimum of 4 million DM for cut flowers (Association of Dutch Flower
Auctions). Some of the larger species, e.g., C. medium and C. pyramidalis,
have traditionally been used as cut flowers, and a number of smaller species, in
particular C. carpatica and C. isophylla, are grown as pot plants. On a smaller
scale, a wide range of species are used as perennials and by alpine gardening
enthusiasts.

1 Research Group for Plant Breeding and Propagation, Department of Ornamentals. Danish
Institute of Plant and Soil Science, Kirstinebjergvej 10, DK-5792 Arslev, Denmark
2 Department of Applied Biological Sciences, Faculty of Agriculture, Saga University. 1 Honjo,
Saga 840 Japan

Biotechnology in Agriculture and Forestry. Vol. 41


Medicinal and Aromatic Plants X (cd. by Y.P.S. Bajaj)
Springer-Verlag Berlin Heidelberg 1998

46

K. Brandt and K. Ishimaru

Fig. 1. Flowering plants of Campanula isophylla (photograph H. Justesen)

1.2 Conventional Utilization

The plants used in folk medicine were collected from nature. Drugs were made
from roots or from the above-ground parts of flowering plants, that were dried
and prepared as broths or infusions (Gastaldo 1978; Barnaulov et al. 1983).
The roots and leaves of one species, C. rapunculoides, can be used as a
vegetable (Becker-Dillingen 1950). No information is available regarding
commercial production of drugs from Campanula. A number of species
have been investigated for their contents of metabolites (Table 1) of possible
pharmacological importance. In addition, one paper states the isolation
of two alkaloids, campedin and (-)lobeline, from C. medium (Dapke and
Fritsch 1970).
In many of these investigations the plant material consisted of mixtures of
plant organs, often all epigeal parts of flowering plants. However, at least the
contents of flavonoids, including anthocyanins, and phenolic acid derivatives
show dramatic differences when comparing leaves, stems, developing flower
buds, and petals (Fig. 2). Also for inulins and polyacetylenes, differences were
found when more than one plant part was tested (Pollard and Amuti 1981;
Tada et al. 1995a). Therefore, some investigations should be considered only
as a guideline to the compounds present in all tissues of the plant, since precise
information on the relative proportions of flowers, leaves, stems, and flower
buds is not available.
Campanula species contain many of the secondary compounds that are
the active principles in plants used for medicinal purposes. Generally, the

47

Campanula (Bellflower) Species

Anthocyanins ~ Flavonoids

i3 Phenolic acids I

0000
0000

~:i!.

1000

100
40

.~.

""

'"

<lJ

:>
ell
<lJ

....l

l!

'"
S

.....<lJ

Vl

Ii

'"

'"d

;:j

..0

...,

<lJ

C. isophylla

'"

~
.....
<lJ

p.,

I ri
'"

<lJ

:>

ell
<lJ

....l

'"
S

<lJ
.....

Vl

'"

'"d

;:j

..0

...,

.$

.....ell<lJ

Il.;

<lJ

C. rotundifolia

Fig. 2. Contents of anthocyanins, flavonoid glycosides, and phenolic acid derivatives in C.


isophylla and C. rotundifolia: fully expanded leaves; stems (2-15 cm above the base of the plant);
flower buds (at least 3 days prior to anthesis); and petals from open flowers. Measured in
EtOH: H 20: formic acid extracts as the sum of all compounds with absorption spectra typical of
each class of compounds. Within each class of compounds identical letters denote samples where
retention time of the major compound was the same. (K. Brandt, unpub\.)

compounds shown in Table 1 or closely related ones are also found in genera
of more commonly utilized medicinal plants such as Platycodon, Codonopsis,
and Adenophora, (e.g., Pollard and Amuti 1981; Goto et al. 1983; Inada et al.
1992; Tada et al. 1995a; Wang et al. 1995). Campanula is thus a genus with a
possible unexploited potential as source of medicinal secondary metabolites,
as pointed out by Barnaulov et al. (1983) and Tada et al. (1996). It may be
fruitful in a survey among Campanula species to search specifically for additional compounds known to occur in, e.g., Platy codon grandiflorum (Inada et
al. 1992; Tada et al. 1975). Platycodon grandiflorum is grown on a commercial
scale in China for production of the traditional drug Jiejeng, in Japan (Kikyo)
and in Korea (Doraji) (Hosoda et al. 1992; Kim et al. 1995).
Commercial propagation of Campanula for ornamental purposes is by
seed, division, or cuttings. Biannual species, such as Campanula medium and
C. pyramidalis, are seed-propagated, since the plant usually dies after
flowering. In addition to seeds, the perennial species, e.g., C. isophylla and C.
carpatica, are propagated by division or cuttings depending on the growth
habit of each species.
Although micropropagation of Campanula is relatively easy, it is used
only to a small extent, since this propagation method is economically competitive only in special cases, e.g., to propagate disease-free elite stock plants,
initial multiplication of new cultivars, or clonal propagation of biannual
species (Brandt 1997).

48

K. Brandt and K. Ishimaru

Table 1. Overview of studies on metabolites in Campanula


Species

Reference
Flavone"
Coumarin 3a
Sterol"
Anthoc. 7d
Inulin 6,
Polyac. 8b
Flavonol"
Triterpene"

C. alliariifolia

C. bayerniana
C. betulaefolia
c. bononiensis

Q
Q

C. choziatowskyi
C. carpatica

C. cochleariifolia
C.
C.
C.
C.

C.
C.
C.
C.
C.

moesica
oblongifolia
ochroleuca
ossetica
patula

C. persicifolia

C,M,B'

+
C

+
M,C,B'

Q
Q

+
C

+
+

+
+

+
+
+
C,R

+
+
+

+Q,K f

C. punctata

+
V

C. punctata var. hondoensis

C. rapunculoides

+
+
+

C. phyctidocalyx
C. poscharskyana

C. raddeana

C. kirpichnikovii
C. kolenatiana
C. latif/ora
lactiloba
leskovii
letschschumensis
makaschvilii
maleevii
medium

hypopolia
isophylla
istriaca
kemulariae

C.
C.
C.
C.
C.
C.

crystallocalyx
dolomitica
elegantissima
glome rata

C. grossekii
C.
C.
C.
C.

+
Q

+
+

+
M

Dzhumyrko (1984); Christensen


(unpub!.)
Dzhumyrko (1985)
Pollard and Amuti (1981);
K. Brandt (unpub!')
Dzhumyrko (1985)
Pollard and Amuti (1981);
Brandt et a!. (1993)
Pollard and Amuti (1981);
Christensen (unpub!')
Lam and Kaufmann (1969)
Dzhumyrko (1984)
Dzhumyrko (1985)
Pollard and Amuti (1981); K.
Brandt (unpub!');
Tada et a!. (1995a)
Pollard and Amuti (1981);
Lam and Kaufmann (1969)
Dzhumyrko (1974)
Brandt et a!. (1993)
Stanic et al. (1989)
Dzhumyrko (1985);
K. Brandt (unpub!.)
Dzhumyrko (1984)
Dzhumyrko (1985)
Pollard and Amuti (1981);
L.P. Christensen (unpub!.)
Lam and Kaufmann (1969)
Dzhumyrko (1984)
Teslov and Podushkin (1988)
Terahara et a!. (1990);
Tada et a!. (1995a, 1996)
Lam and Kaufmann (1969)
Dzhumyrko (1984)
Dzhumyrko (1985)
Teslov and Blinova (1974);
Teslov (1980b); Pollard and
Amuti (1981)
Teslov and Koretskaya (1983);
Teslov (1984, 1986, 1988, 1990);
Pollard and Amuti (1981);
Tada et a!. (1995a)
Lam and Kaufmann (1969)
Pollard and Amuti (1981);
Brandt et al. (1993)
Pollard and Amuti (1981);
Tada et al. (1995a)
Lam and Kaufmann (1969);
Tada et a!. (1995a)
Dzhumyrko (1985); Pollard
and Amuti (1981); Lam and
Kaufmann (1969)
Pollard and Amuti (1981);
K. Brandt (unpub!')

Campanula (Bellflower) Species

49

Table 1. Continued
Species
C. rotundifolia

C.
C.
C.
C.

takhtadzhianii
thyrsoides
trachelium
vidalii

Flavone l '
Coumarin"
Sterols"
Anthoc. 7d
Reference
Flavonol"
Triterpene'"
Inulin 6'
Polyac Sb

+ K

+ M

+
+
+

Teslov (1980a, 1981);


Teslov et a!. (1983); Pollard
and Amuti (1981); K. Brandt
(unpub!'); Tada et a!. (1995a)
Dzhumyrko (1985)
Pollard and Amuti (1981)

Pollard and Amuti (1981);


L.P. Christensen (unpub!.)

Luteolin glycosides; 2 Quercetin (Q) and kaempferol (K) glycosides; , Fraxetol and its glycosides;
Ursolic acid; .\ Sterols; 6 Inulin oligomers; 7 Anthocyanins: bisdeacylplatyconin (B), campanin (C),
monodeacy1campanin (M), rubrocampanin (R), violdelphin (V); 8 polyacetylenes.
Notes:
, In epigeal parts of flowering plants.
h In leaves.
, In stems.
J In flowers.
, Different chemotypes.
f Compounds found together in one sample.
1

2 In Vitro Approaches
2.1 Review of Tissue Culture Studies

There are only a few reports on tissue culture of Campanula (Table 2).
Micropropagation of Campanula using shoot clusters was reviewed by Brandt
(1997), based on earlier work (Brandt 1992, 1994), and here flower bud culture
was mentioned as a possible tool for studies of anthocyanin accumulation.
Production of secondary metabolites (the polyacetylene lobetyol and its
glucosides) from hairy root cultures of C. medium has been reported recently
(Tada et al. 1996).
Since Campanula is not an economically important medicinal plant,
both known cases of in vitro production of secondary metabolites are
described by the respective workers as model systems for biosynthetic
studies. The contents of secondary metabolites differ among various kinds
of plant tissue (Fig. 2), so it is important to be able to grow several plant
organs in vitro in order to investigate all the processes taking place in the
plant.
2.2 Establishment of Tissue Cultures and Micropropagation

Unless specifically mentioned, the following refers to cultures grown at 24C


with a photoperiod of 16h day-land an irradiance of20 .umolm-2s-1 (inside the
culture vessels) from cool white fluorescent tubes.

K. Brandt and K. Ishimaru

50

Table 2. Summary of in vitro culture studies on Campanula species

Species

Purpose of study

Observationslremarks

Reference

C. isophylla

Development
of methods for
micropropagation

Use nodes with small buds for


initation, and basal part of shoot
clusters on 1-2mg/l BA for propagation.
Omit growth regulators for rooting,
genetic variation is important for both
proliferation and rooting

Brandt

Analysis of
genetic variation
in formation of
shoots and roots

Within clones, clusters with large


shoots have many roots and few shoots.
Among clones, size and number of shoots
were not correlated, but root number still
depended on shoot size. So selection of
clones with many shoots will not
compromise rooting ability

Brandt

Polyacetylene
production in
hairy root
cultures

Agrobacteriun rhizogenes induced hairy


root cultures on sterile leaf disks. In
liquid medium MS without hormones was
superior to other formulations regarding
both biomass and polyacetylene production

Tada et al.

Review of
published studies
and other
observations

In vitro requirements generally differ little


among species. Important to keep cultures
vegetative. Flowers may develop in vitro,
particularly if explants are taken from
generative shoots

C. isophylla

C. medium

C. isophylla
C. carpatica
C. pyramidalis

(1992)

(1994)

(1996)

Brandt
(1997)

Fig. 3. Shoot culture of C. isophylla after six subcultures on MS with 1 mg/l BA. (Photograph K.
Brandt)

2.2.1 Shoot Cultures

Shoot cultures are initiated from vegetative buds (Fig. 3). The best explant
type is a 6-mm stem segment with a 1-2-mm axillary bud, taken from a position
not in direct contact with the soil surface. The stem segment is surfacesterilized in two separate steps, alternating with trimming of the cut ends

Campanula (Bellflower) Species

51

(Brandt 1992). The final explant, retaining about 2mm of the stem, is cultured
on MS medium (Murashige and Skoog 1962) with the macronutrients reduced
to 50%, 30gl- 1 sucrose, 4.4,uM benzyladenine (BA), and 6gl- 1 agar (Difco
Bacto). This procedure was used for C. isophylla and C. pyramidalis. For C.
carpatica 12 g 1-1 agar was used, and a photoperiod of 12 h day -I. After 5 weeks
on initiation medium, the explants were moved to a propagation medium with
full-strength MS salts, but otherwise identical to the initiation medium. Subsequently, every 5 weeks, all shoots were excised 3mm above the basal part of
the shoot cluster, using the basal parts for further culture. The shoot tips are
discarded, or they can be transferred to rooting medium (hormone-free, halfstrength MS) for production of plantlets.
Shoot cultures have not been used for deliberate production of secondary
compounds. However, it was observed that if the shoot cultures are grown on
media with high concentrations of sucrose (to facilitate root formation; Brandt
1997), the leaf color became more reddish, indicating an enhanced accumulation of anthocyanin.
2.2.2 Flower Bud Cultures

The procedure is similar to shoot cultures, but the explant is instead taken
from a generative bud (Fig. 4). On a flowering plant, the buds will be more or

Fig. 4. Flower bud culture of C. isophylla 2 months after initiation. Generative explants were
grown on MS without hormones, with 2% sucrose and an irradiance of 35 ,umol m -2 S-I. (Photograph K. Henriksen)

52

K. Brandt and K. Ishimaru

less generative according to the position on a shoot; the closer to the apex, the
more generative are the buds. Positions where the meristems are generative
are characterized by small, lanceolate sepal-like leaves, readily distinguishable
from the larger, usually rounded leaves on vegetative parts of the plant (refer
to Fig. 1). Explants were subcultured when the petals of the first visible flower
bud reached a length of 2 mm.
Using this procedure similar results are obtained in C. isophylla and in C.
carpatica. In C. isophylla, in vitro flowering has been obtained only using this
procedure, while in C. carpatica formation of flower buds was observed also in
previously vegetative shoot cultures cultured in vitro in continuous light (K.
Brandt, unpubl.). The development of the flower buds depends on both media
and irradiation. Even with optimal combinations of the factors tested so
far, most explants still revert to vegetative growth. However, the experimental
system is now so well developed that it can be used to study the synthesis of flower compounds in an isolated system with a highly controlled
environment.
2.2.2.1 Growth Regulators

The effects of adding the growth regulators BA and gibberellic acid (GA3) to
bud cultures of C. carpatica were investigated (Fig. 5). It appears that both
growth regulators depress the percentage of buds that develop into flowers, so
subsequent experiments were done without growth regulators.
2.2.2.2 Sucrose and Irradiance

The effects of different sucrose concentration in the medium and the irradiance given to flower bud cultures of C. isophylla differed during the progression through the culture period. The media were prepared with four levels of

40,~
~30
o

'-'
[fJ

20

(~

510

\)

o - ~
o 1

234
BA concentration, mg/l

Fig. 5. Effects of BA (0.5--4 mg/I) and GA3 (0 or 0.5 mg/I) on in vitro flower development in C.
carpatica. (J.M. Lanka, unpubl.)

Campanula (Bellflower) Species

53

Fig.6. Percentage of surviving uncontaminated explants of C. isophylla. Survival was recorded as


explants with active growth of leaves or flower buds 5 weeks after culture initiation

Fig. 7. Percentage of surviving explants of C. isophylla that completed development of at least


one flower

sucrose: 0, 2, 4, and 6%. Mannitol was added to some of the media to compensate for the differences in osmolality, thus the concentrations of mannitol were
respectively 3.15, 2.1, 1.05, and 0%. In the initial stages, only sucrose concentration affected the survival of the explants (Fig. 6). The experiment showed a
well-defined optimum at 2% sucrose in the medium, irrespective of the irradiance. The effect of sucrose in the medium was highly significant (p < 0.0001),
while the irradiance had little effect on explant survival.
For the subsequent development of the explants into flowering or
nonflowering shoots, an interesting interaction between sucrose and irradiance appeared (Fig. 7).
The lower the irradiance, the more sucrose was required in the medium to
support the continued development of the meristem into a flower. No flowers
developed without sucrose, irrespective of the irradiance. It is known that light
can promote flower formation, but this has usually been attributed to effects

54

K. Brandt and K. Ishimaru

on either flower induction or carbohydrate availability (Bernier et al. 1993). In


the present system all buds are already induced, and sucrose is supplied in
excess in some of the media; light still appears to playa crucial role in determining how many of the buds will develop into flowers. While the percentage
of explants capable of forming flowers differed strongly among the treatments,
the weight of each flower showed no significant variation.
2.2.3 Genetic Transformation and Hairy Root Cultures

A method for infection with Agrobacterium rhizogens and establishment of


hairy root cultures of C. medium was described by Tada et al. (1996), and
secondary metabolites studied.
Seeds were surface-sterilized for 8 min in 2% NaOCI with Tween 20 and
aseptically germinated on half-strength MS salts solidified with 0.25% Gelrite
at an irradiance of 60.umolm-2s-1. Leaf disks from the axenic plantlets were
infected with Agrobacterium rhizogenes A13 strain harboring the binary vector
plasmid pBI121 + wild mikimopine-type Ri (Otani et al. 1993).
The bacteria were eliminated with 0.5 gl-l Claforan, the hairy roots transferred to liquid MS without hormones (SOml per 100-ml Erlenmeyer flask) and
maintained in the dark on a rotary shaker (100rpm) (Fig. 8). One clone with
good growth was selected and used for transfer to other media. Transformation was confirmed by detection of NPT II and GUS genes by PCR (Fig. 9;
Otani et al. 1993).
Transformed cultures were inoculated in five hormone-free media and
grown as before for a 4-week growth period: MS, 112 MS, BS (Gamborg et al.
1968), WP (Lloyd and McCown 1980), and RC (Thomas and Davey 1982).
Performance was best on MS, BS, and WP, and these media were chosen for
further studies.

Fig. 8. Hairy root induction of C. medium, infected with Agrobacterium rhizogenes AI3 by the
coculture method. (Photograph K. Ishimaru)

Campanula (Bellflower) Species

55
peR
analysis
tor detecting rol

and

GUS

genes

225 0 b

1 1 05b

M PC PC C C T T M
I

ra l

CUS

'I'

CUS

M
; 1 00 ba se I adder lIa rker
C - ro l ;Positive contr o l
PC - GUS;P osit i ve contro l
C - ro I
;Non -t ransforllant
C - GUS
; Non-transtorlla nt
T
;Haory roots
p

Fig. 9. peR analysis for detecting rol and GUS genes. (Photograph H. Tada)

Fresh hairy roots (ca. 200 mg) were inoculated into the three media (Fig.
10). The hairy roots were harvested weekly for 8 weeks and the dry weights
recorded. MS supported the best growth, mostly because the cultures started
to grow soon after transfer.
The dry weight of the roots rapidly increased until the end of the culture
time (8 weeks) to reach the maximum amount (562.3mg per flask) (Fig. 11).
In contrast, a lag time of more than 5 weeks was observed in the two other
media. The maximum root mass in B5 and WP, at week 8, were almost half of
that in MS. The formation of polyacetylenes in hairy roots is discussed in
Section 2.3.3.
2.2.4 Callus and Suspension Cultures

No publications are available on deliberate induction of callus in Campanula.


However, callus formation was reported as an unintended event, in micropropagation of C. isophylla (Brandt 1992). When naphtaleneacetic acid

56

K. Brandt and K. Ishimaru

Fig. 10. C. medium hairy roots cultured in hormone-free MS (left) , B5 (center) , and WP (right)
liquid media for 4 weeks in the dark. (Photograph K. Ishimaru)

600
~ 500

- 0-

5...

400
.c
!)I)
.Qi
~

t'

MS
B5
WP

300
200
100
0

4 5
Weeks

Fig. 11. Growth (mg/ftask) of C. medium


hairy roots cultured in MS, B5, and WP liquid media (bars represent standard errors).
(Tada et al. 1995a)

(NAA) was added to MS medium at a concentration of O.S4.uM, 91 % of


shoot clusters formed a soft, white, rather friable callus. The callus
formed mainly around the base of the shoot cluster, but also the roots were
thickened and malformed, and appeared on the way to be transformed into
callus.
Suspension cultures have been tested as a method for propagation
of C. pyramidalis (D. Holdgate, pers. comm.); the technique was successful,
but the experiments were terminated because the plantlets produced were too
expensive. Friable callus was produced using 2,4-D, and somatic
embryogenesis was induced by removing the auxin (D. Holdgate, pers.
comm.).

Campanula (Bellflower) Species

57

2.3 Extraction and Structures of Anthocyanins and Other


Secondary Metabolites

2.3.1 Anthocyanin Extraction and Structures

Anthocyanin content of flowers developed in vitro was determined by HPLC


as described by Brandt et al. (1993), and adapted for small samples (less than
1 g) by Justesen et al. (1997). Whole flowers (including ovaries) were harvested
aseptically, since the remaining shoot would sometimes generate another
flower a few weeks later. Fresh weight was determined before freezing at
- 20C. The flowers were extracted separately by homogenizing with 2 ml cold
MeOH-H 20-HC0 2H (10:7:3) plus 4ml cold MeOH-H 20-HC0 2H (10:3:3)
per g frozen plant material. HPLC analysis was carried out on a RP-18 column
with solvent A = H 20-HC0 2H (19: 1), B = MeOH-H20-HOAc (10:7:3), and
a gradient starting with 0% B, 10% B at 3min, 25% B at 8min, 60% B at
22min, and 100% B at 25 min. Using a diode array detector to separate the
phenylpropanoid classes according to absorbtion spectra, anthocyanins were
detected at 535 nm, flavones at 360 nm.
The five major anthocyanins found in the Campanula species examined
so far are shown in Fig. 12. Campanin, rubrocampanin, and monodeacylcampanin are known only from Campanula (Terahara et al. 1990; Brandt et al.
1993). Bisdeacylplatyconin was described first as a degradation product of
platyconin from Platycodon grandifiorum (Goto et al. 1983) and violdelphin
was identified in Delphinium hybridum (Kondo et al. 1990). Both were later
found to occur also in Campanula (Brandt et al. 1993). Genotypes containing
rubrocampanin as the major anthocyanin have pink flowers (Terahara et al.
1990), and mutants where the biosynthesis is blocked after bisdeacylplathyconin have a very pale, light purple color (Brandt 1990). Flowers
of chemotypes with the acylated delphinidin derivatives campanin, monodeacylcampanin, and violdelphin all have purple-blue colors with the same
hue; they cannot be distinguished without a chemical analysis (Brandt 1990; K.
Brandt, unpubl.).
The biosynthetic sequence of the wild type (Type C) has been found by
examination of the genetics and of the compounds found in genotypes defective in some of the steps shown in Fig. 12 (Brandt et al. 1993).
2.3.2 Investigation of Anthocyanin Accumulation

An important qualitative character in Campanula flowers is the proportion


of bisdeacylcampanin, since this is an unacylated form, unable to take part
in intramolecular copigmentation (Brouillard 1988) and therefore not contributing to the flower color (Brandt et al. 1993). Bisdeacylplatyconin is
the major anthocyanin in developing flower buds. The concentration of
bisdeacylplatyconin peaks 3 days prior to anthesis, where a conversion to the
acylated compounds sets in, then decreases exponentially over a period of 10
days (Justesen et al. 1997). Knowledge of the course of biosynthesis of

K. Brandt and K. Ishimaru

58

Bisdeacylplatyconin

Violdelphin
ID~

~O

1D~~o
OIl

OIl

ID~-=-O

ID~O
OH

ID

Monodea:rlcampanin

1D~~o
OH

1D~~o
OIl

OIl

ID~-=-O

ID~O
OIl
ID

Fig. U. The major anthocyanins found in Campanula and the proposed biosynthetic sequence of
the three compounds found as major anthocyanins in wild-type flowers (cf. Table 1).
Bisdeacy1campanin is a minor component in petals of all genotypes analyzed, and is the major
anthocyanin in light-blue-flowering mutants of C. isophyUa and C. carpatica. Rubrocampanin has
been found only in red-flowering mutants of C. medium. (After Brandt et al. 1993, with kind
permission from Elsevier Science Ltd., The Boulevard, Langford Lane, Kindlington OXS 1GB,
UK; and Terahara et al. 1990)

acylated anthcyanins is relevant for the quality of pot plants: a pale color in the
new flowers formed after a pot plant is taken from the nursery to a shop or the
home of the customer is a well-known problem for retailers and growers of pot
plants. This is a question of both quantity and quality of the anthocyanins: not
only is the total concentration of anthocyanins in the flowers reduced, also the
proportion of bisdeacylplatyconin relative to the acylated anthocyanins is

59

Campanula (Bellflower) Species

Campanin

OH

Rubrocampanin

Fig. 12. Continued

greater in plants grown with insufficient light (Brandt 1990). Experiments with
intact plants showed that while the total concentration of anthocyanin was the
same, flowers of type B (with the diacylated monodeacylcampanin as the end
product) accumulated less bisdeacylplatyconin during development and processed it faster into the acylated compounds than type C flowers (able to
accumulate the triacylated campanin) (Justesen et al. 1997). This indicates that
the relative composition of anthocyanin (conversion of one anthocyanin into
another) may be easier to manipulate than the total concentration (the
number of anthocyanin molecules produced).
Efforts to improve the quality require a thorough understanding of the
effects of insufficient light on anthocyanin biosynthesis. It is particularly important to understand if it is due to a direct influence of light on the developing
bud, e.g., on the induction of acylating enzymes, or if it is an indirect effect, due
to starvation for carbohydrates, the products of photosynthesis. The in vitro
flower bud cultivation system described above was established in order to
make this kind of investigation possible. The idea was to develop an experimental system where the effects on anthocyanin accumulation of light and of
availability of photosynthate could be varied independently.

60

K. Brandt and K. Ishimaru


Fig. 13. In flowers developed in
vitro on three sucrose concentrations and with three irradiances

~ 15,,~--.------.----~

'-'

'2

8 1O +----'~+---f------l

Type B
o TypeC

....n:l>.

0..

~ 5 +---~~-r--~
v

n:l
<J.)

'"0
Cfl

Q5

20

40

60

Fig. 14. In flowers of the chemotypes Band C, containing monodeacylcampanin and campanin as
the end product of biosynthesis,
respectively. (K. Brandt, unpub!.)

Figs. 13,14. Percentage of total anthocyanin of the nonacylated anthocyanin bisdeacyl-platyconin


in flowers of C. isophylla developed in vitro

An experiment was done with genotypes of C. isophylla that were seedlings from the same cross, segregating for the genes that control the acylation
of monodeacylcampanin to campanin. The seedlings were classified according
to the presence or absence of campanin as major anthocyanin in flowers, into
the phenotypes type C and type B. (Fig. 12). One result of the first experiment
is shown in Figs. 13 and 14.
Both irradiance and sucrose availability influence the acylation of
the anthocyanins; however, to some extent they appear to be able to substitute
for each other: variation in irradiance has greatest effect when sucrose is low,
and vice versa. Sucrose concentrations of 4 and 2 % are not so low that
the effect of light can be explained simply as the result of photosynthesis.
Rather, the results indicate a general requirement for light, but that this
requirement disappears when the sucrose supply is superoptimal. Corresponding to the situation in vivo, the percentage of bisdeacylplatyconin in

61

Campanula (Bellflower) Species

in vitro flowers is consistently higher in type B flowers than in flowers of


type C.

2.3.3 Polyacetylene Extraction and Structures


Polyacetylene contents of dried samples of the hairy roots were measured on
HPLC as described earlier for Lobelia inflata by Ishimaru et al. (1993).
Lyophilized hairy roots (20-30mg) were crushed by pestle and extracted
with MeOH (2 ml) for 15 h at room temperature. After filtration with
Millipore filter, the extract was injected (3 pI) to HPLC: column, Inertsil
ODS (4.6mm i.d. X 250mm); mobile phase, MeCN-H 20 (1:4 ~ 9: 1, linear
gradient in 30min); flow rate, 0.68ml/min; detection 270nm (UV); column
temperature 40C; R t (min): lobetyolinin (15.9), lobetyolin (19.8), and
lobetyol (23.9).
The structures found in C. medium are shown in Fig. 15. In a recent survey
(Tada et al. 1995a), these three compounds were found to be widely distributed in the Campanulaceae. Apart from Campanula (cf. Table 1) they were
shown to be present in the genera Adenophora, Lobelia, Platycodon,
Specularia, and Wahlenbergia.

2.3.4 Investigation of Polyacetylene Accumulation


In all media (cf. Sect. 2.2.3), the main polyacetylene constituent was lobetyolin
(monoglucoside of lobetyol). Both the maximum concentrations and the
rates of accumulation and decline differed among the three media (Fig. 16).
The maximum contents of lobetyolin in percent of dry weight observed in
these media (3.74 in MS at week 6, 2.69 in B5 at week 4, and 3.13 in WP at
week 7) were over 130 times higher than the 0.02 found in leaves of the intact
plant (Tad a et al. 1995a). The specifically high content of lobetyolinin
observed in C. medium hairy roots was higher than that found in corresponding hairy root cultures of the medicinal plant Platycodon grandiflorum
(0.41 %; Tada et al. 1995a) and comparable to that found in four species of the
genus Lobelia (Ishimaru et al. 1993, 1994; Tada et al. 1995b). The same
experimental setup has been used to study the effects of light or darkness
H

I
I
Me-C=C-C=C-C=C-CH-CH-C=C-CH CH CH OH
I
I I
I
2

OH OR

lobetyol : R=-H
lobetyolin : R=-gIc
lobetyolinin : R=_glc 6 _lgIc
Fig. 15. Structures of polyacetylenes. (Tada et al. 1995a)

62

K. Brandt and K. Ishimaru


4.0

-0-

lobetyol

4.0

-0-

3.5

--_D

lobetyolin
lobetyolinin

3.5

- - lobetyolin
---fr- lobetyolinin

~3.0

.~ 2.5

~2.0
~1.5

e}'.

1.0

0.5

-=

O!)

1:

O!)

4.0

-0-

3.5

--

3.0

.~ 2.5

-0

2.0

~ 1.5

1.0

0.5

O~

lobetyol

345
Weeks

678

f.l

f.!
2

Z!
3

f.:-=f,l

J~!

Weeks

."7

"
8

lobetyol

3.0

.~ 2.5

C 2.0

-0

~ 1.5
e}'.

1.0

0.5
O+-~~~~~~~~~~~

234

Weeks

678

Fig. 16. Contents of polyacetylenes in hairy root cultures of C. medium cultured in different
media. (Tad a et al. 1995a)

during culture on the accumulation of polyacetylenes in P. grandiflorum (Tada


et al. 1995a).

3 Conclusion and Prospects


Plants of the genus Campanula are used mostly as ornamentals. However,
a number of species have been used in folk medicine, and as far as is
known the contents of secondary metabolites are similar to species with
established medicinal properties from other genera, e.g., Platycodon
grandiflorum.
Two kinds of in vitro systems were established for investigations of accumulation of secondary metabolites by growing specific plant organs in isolation. These are flower bud cultures of C. isophylla and C. carpatica for
investigations of anthocyanins, and hairy root cultures of C. medium that
accumulate polyacetylenes. In both cases, the results have implications for
understanding the control of biosynthesis of secondary metabolites, in both

Campanula (Bellflower) Species

63

tissue culture and the physiology of intact plants. The genus is relatively easy
to cultivate in vitro.

4 Protocol
4.1 Flower Bud Culture
1. Culture Initiation. From the generative part of a flowering stem. a 6-mm segment with a 1-2mm axillary bud is surface-sterilized with Korsolin or NaOCI solution in two separate steps.
alternating with trimming of the cut ends to a final length of about 2 mm. For each species, the
same salts, vitamins, and gelling agent as for micropropagation are used (Brandt 1997), and no
growth regulators. Photoperiod must be 16 h day-lor longer. For initiation of bud growth 2-3%
sucrose is optimal, but 4-6% sucrose results in a larger percentage of flowering explants.

2. Production of Flower Buds. Explants may be subcultured one or more times. Most primary
flowers are produced 6 to 10 weeks after culture initiation.
3. Anthocyanin Analysis. Flowers are extracted by homogenizing with 2ml cold MeOH-H 20HC0 2H (10:7: 3) plus 4ml cold MeOH-H20-HCO,H (10:3: 3) per g frozen plant material. HPLC
analysis is carried out on a RP-1S Licrospher column (4.6mm i.d. X 250mm) with solvent
A = H 2 0-HC0 2 H (19: 1), B = MeOH-H2 0-HOAc (10: 7: 3). The column is equilibrated with
solvent A for 10 min before injection of up to 100,u1 extract. Elution is on a gradient with 10% B
at 3 min, 25% B at Smin, 60% B at 22 min, and 100% B at 25 min, continuing for 10min. Column
temperature 35 DC. Anthocyanins are detected at 535 nm. R, (min): bisdeacylplatyconin to.S,
monodeacylcampanin 14.2, camp an in 17.S, and violdelphin IS.7.

4.2 Hairy Root Culture


1. Culture Initiation. Seeds are surface-sterilized for Smin in 2% NaOCI with Tween 20 and
aseptically germinated on half-strength MS salts solidified with 0.25 % Gelrite at an irradiance of
60 ,umolm-' S-I. Leaf disks from the axenic plantiets are infected with Agrobacterium rhizogenes
An strain harboring the binary vector plasmid pBI121 + wild mikimo-type R i .

2. Production of Hairy Roots. The bacteria are eliminated with 0.5 g 1-1 Claforan, the hairy roots
transferred to liquid MS without hormones (50ml per 100-ml Erlenmeyer flask) and maintained
in the dark on a rotary shaker (100 rpm). Transformation is confirmed by detection of NPT II and
GUS genes by PCR.
Transformed cultures are inoculated in hormone-free MS medium or other media and grown
as before for 4-S weeks (on MS 6-week growth periods give the highest yield per flask). At
subculture, fresh hairy roots (ca. 200 mg) are inoculated into, each flask.
3. Polyacetylene Analysis. Lyophilized hairy roots (20-30mg) were crushed by pestle and
extracted with MeOH (2ml) for I5h at room temperature. After filtration with MiIIipore filter,
the extract was injected (3 Ill) to HPLC: column, Inertsil ODS (4.6mm i.d. X 250mm); mobile
phase, MeCN-H 20 (1:4 ---7 9:1, linear gradient in 30 min); flow rate, 0.6Sml/min; detection
270 nm (UV); column temperature 40 DC; R, (min): lobetyolinin 15.9, lobetyolin 19.5, and lobetyol
23.9.
Acknowledgments. This work was supported in part by the Danish Agricultural and Veterinary
Research Council (grant no. 13-4559) The authors acknowledge the contributions from the

64

K. Brandt and K. Ishimaru

students H. Tada. H. Justesen and J.M. Lonka, and wish to thank K. Henriksen and M. Hansen for
expert technical assistance.

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V Catharanthus roseus (Periwinkle): In Vitro


Culture, and High-Level Production of Arbutin by
Biotransformation
M. YOKOYAMA and S. INOMATA

1 Introduction
Catharanthus rose us (family Apocynaceae) is grown as an ornamental plant in
many countries, although it originated from Madagascar. It is also known as
Madagascar periwinkle or Cape periwinkle. This plant was used traditionally
as a crude medicine for diabetes and other ailments. It has also been used as
a substitute for hops in brewing beer. Now, however, C. roseus is most useful
as a source of various alkaloids; approximately 90 indole alkaloids have been
isolated from it, the most valuable being the dimeric alkaloids vinblastine and
vincristine, which show antitumor activity. They are very similar in chemical
structure, but their activity spectra and side effects are extremely different:
vinblastine is effective against Hodgkin's disease, choriocarcinoma, and the
like, while vincristine is mainly employed to treat childhood acute leukemia.
Vinblastin shows bone marrow toxicity, whereas vincristine is toxic to the
nervous system. Due to the very low yields of these dime ric indole alkaloids in
the plant (approx. 0.0005%), attempts have been made to produce alkaloid
and other secondary metabolites in cell and tissue cultures. General reviews of
work in this field have been published (see Heijden et al. 1989; David and
Tempe 1993; Hirata et al. 1994; Sakurai and Fujioka 1996).
In this chapter, attention is focused on another attribute of C. roseus cells
in culture, i.e., the ability to biotransform exogenously added hydroquinone
(HQ) to arbutin. We have extensively examined the feasibility of efficient
production of arbutin using C. roseus cell culture (Yokoyama et al. 1990, 1996;
Inomata et al. 1991).
Plant cell cultures catalyze a wide range of biotransformations, such as
glucosylation, glucosyl esterification, hydroxylation, oxidation-reduction
between alcohols and ketones, reduction of carbon double bonds (C=C),
hydrolysis, isomerization, epoxidation, dehydrogenation, methylation,
demethylation, and others (Furuya 1988; Suga and Hirata 1990; Yokoyama
1996). However, major biotransformations by plant cell cultures are
hydroxylation and glucose conjugation involving glucosylation and glucosyl
esterification; these reactions are the subject of 45 and 41 % of published

Pharmaco Science Research Laboratories, Shiseido Research Center, 1050 Nippa-cho, Kohokuku, Yokohama 223, Japan

Biotechnology in Agriculture and Forestry, Vol. 41


Medicinal and Aromatic Plants X (ed. by Y.P.S. Bajaj)
Springer-Verlag Berlin Heidelberg 1998

68

M. Yokoyama

reports in this field, respectively (Yokoyama 1996). Hydroxylation reactions


are catalyzed by cytochrome P-450 in many cases. For example, biotransformation of digitoxin to digoxin by 12 fJ-hydroxylation using Digitalis lanata
cell culture is cytochrome P-450-dependent (Petersen and Seitz 1985), and
biotransformation of tryptamine to serotonin is also believed to be cytochrome PA50-dependent (Courtois et al. 1988). Since cytochrome PA50 is
well known to participate in detoxification of drugs, hydroxylation may be
regarded as an initial step in detoxification. Glucosylation at the resultant OH
moiety may proceed as a subsequent step in detoxification, in plants.
Microorganism-mediated biotransformation also produces glycosides in
some cases, but such glycosides are not major products, and glucosides are not
known as secondary metabolites in microorganisms. Animals generally seem
to use glucuronylation reactions for detoxification. Thus, glucosylation and
hydroxylation seem to be rather specific biotransformations of higher plant
cells, and the high-yield biotransformations which have been reported so far
in plant cell cultures have mainly involved prduction of glucosides and
hydroxylated substances (Table 1).
Arbutin (Fig. 1), the glucosylated derivative of HQ, is the main active
component of Arctostaphylos uva-ursi Sprengel, which was used as a urethral
disinfectant for many years before antibiotics were developed. This glucoside
is also a potent suppressor of the synthesis of melanin in human skin
(Akiu et al. 1988), without apparent side effects (ltabashi et al. 1988). The

OH

Fig. 1. Structure of arbutin

OH

Table 1. High-yield production by biotransformation

Product

Substrate

Type of
reaction

Plant species

Yield
(gil)

Reference

Arbutin

Hydroquinone

Glucosylation

Rauwolfia
serpentina

18

Catharanthus
roseus
Rauwolfia
serpentina
Peganum
harmala
Datura
innoxia

9.2

Lutterbach and
StOckigt
(1992)
Inomata et al.
(1991)
Lutterbach et
al. (1993)
Courtois et al.
(1988)
Umetani et al.
(1982)

p-Hydroxyphenyl- Hydroquinone
O-primeveroside
Serotonin
Tryptamine

Hydroxylation

U mbelliferone

Glucosylation

Skimmin

Glucosylation

5.8
2.5
1.6

Catharanthus roseus (Periwinkle)

69

cosmetic company Shiseido has developed arbutin as a skin depigmention


agent; a product containing chemically synthesized arbutin' has been on sale in
Japan since 1990.

2 Arbutin Production by Biotransformation Using


C. roseus Cell Culture
2.1 Importance of Cell Strain

Although the selection of a superior cell line to produce useful metabolites is


usual (Parr 1989), little is known about differences in bioconversion by different cell lines of the same plant species (Courtois et al. 1988; Tabata et al. 1988).
We examined the glucosylation of HQ to arbutin by using two morphologically different cell lines (CrA and CrB) of suspension-cultured C. roseus cells
of the same origin, and compared the glucosylation in these cell lines in detail
(Inomata et al. 1994).
CrB was derived spontaneously from CrA (Suzuki et al. 1990). Both
strains showed almost the same growth rate, but the cell shapes are very
different. As shown in Fig. 2, cells of CrA were globular, dense, and smaller
than those of CrB, while cells of CrB were cylindrical in shape with welldeveloped vacuoles.
Changes were examined in the glucosyltransferase activity, which
conjugates glucose to HQ, of both cell strains in the exponential growth
phase. The results are shown in Fig. 3. The enzyme activity was not detected in
either of the cell strains before addition of HQ. When HQ was added, the
enzyme was detected within 6h and reached a peak within 24h in both strains.
However, the maximum enzyme activity of CrB was five fold higher than
that of CrA, this value corresponding well with the difference of the final
arbutin yield per cell fresh weight (Fig. 3). Arbutin formation stopped at
around SOh in CrA, while CrB continued to generate arbutin until 70h with
high activity. The CrB cells survived until 70 h, but all of the CrA cells had died
by 50h.
Yamamoto et al. (1986) reported that vacuoles in high-alkaloid-producing
cell lines of Cop tis japonica and Thalictrum minus were larger than those in
low- or nonproducing cell lines. They suggested that development of vacuoles
might be a prerequisite for alkaloid synthesis, to prepare a reservoir for
storage of the alkaloids. In the case of C. raseus cells, we observed that the
cells expanded about two fold with a corresponding expansion of the vacuoles
as the accumulation of arbutin progressed. Thus, the significantly larger
vacuoles of CrB may contribute substantially to the larger productivity of
arbutin in comparison with CrA.
The selection of superior cell strains appears to be a useful means of
improving the production of compounds by biotransformation in plant tissues,
as has already been established for de novo synthesis (Parr 1989).

70

M. Yokoyama

B
Fig.2A.B. Catharanthus roseus cells cultured for 4 days. A CrA strain; B CrB strain. (Inomata et
al. 1994)

2.2 Sugars as Promoters of Arbutin Production

Sugars are utilized as nutrients via the Embden-Meyerhof pathway. It was


found that the addition of sucrose or glucose caused the production of arbutin
to increase greatly, on the basis of both fresh weight of cells and volume of
medium (Fig. 4). However, essentially no sugar was consumed, not even the
0.5% sugar originally present, although addded sucrose was converted to
glucose and fructose by invertase in the cell wall (Fig. 5). The presumption that
sugars do not act merely as nutrients was confirmed by the observation that
sorbitol also has a similar effect on arbutin production.

71

Catharanthus roseus (Periwinkle)


40~---------------------------.

30

T
11--------....
T

>,

"'-'

'5

'';::;

3:

ra

"-

Q)

OJ

"~1
Q)

(f)

,,

,,

20

,,

l-

(f)

"-

;///1

30

"'-'

.L

"'-'

:;:

I-

"OJ

20

"-

OJ

:::'
:t:: x
>, E
0. 10
0

10

'';::;
::J

(f)

U
::J

-e

<t

OJ

cJ

25

50

75

100

Hours after the addition of HQ


Fig. 3. Change in HQ glucosyltransferase activity and arbutin production in flask cultures of the
two strains of Catharanthus roseus after HQ addition. HQ was periodically added to make 4 mM
at intervals of 24 h until the cells ceased to consume the HQ supplied. Solid lines show the enzyme
activity of CrA (0) and CrB (0). Broken lines show arbutin production per cell fresh weight of
CrA (e) and CrB (.). (Inomata ct al. 1994)

sucrose

- 15

101-

- 10
C

:J

:J

a 10 -

.0

.0

en

glucose

/0

0 a
15

10

L-

en

/0

o~~~~~~~~o

01234567
Sugar added ("10)

Fig. 4. Effects of sucrose and glucose on the production of arbutin in cell culture of Catharanthus roseus.
HQ was added to a final concentration of 4mM on
the 7th, 10th, and 11th days of CrA-suspension culture, and production of arbutin was evaluated on the
14th day. Sugars at the indicated concentrations were
supplied once only, on the 7th day. (Yokoyama et al.
1990)

M. Yokoyama

72

0-

::J

QJ

01

'c
'0

E
QJ

'-

'-

d
01

::J

if)

Fig. 5. Sugars remaining in the medium after the


experiment. After completion of the experiment
shown in Fig. 4, on the 14th day, sucrose (_ .. _), glucose (...... ), fructose (_._) , and the total sugar
( - ) in the medium were analyzed. (Yokoyama et
al. 1990)

7
6
5
I.;

3
2
1

0
7
6
5
4

G1Ucose/~,:::

/;/
..... _._.-.e---.-.-._.-. _.

.. --L .. _.I_.-1._ .. L .. _\it-.

234567
Sugar added (%)

Sugars are known to act as hydroxyl radical scavengers. HQ is assumed to


generate superoxide through its oxidation to paraquinone (Yokoyama et al.
1990), and superoxide radicals can react with hydrogen peroxide, which is
formed through the enzymic or spontaneous dismutation of superoxide radical, to form even more injurious species of oxygen, such as hydroxyl radical
and singlet oxygen (Beauchamp and Fridovich 1970; Kellogg and Fridovich
1975; Hassan and Fridovich 1979). Therefore, we examined the effect of sugars
on cell viability, using triphenyltetrazolium chloride (TTC)-reducing ability as
a measure of cell viability. Figure. 6 shows the results for cultures with added
sucrose and the control; the time courses were similar up to 48h, but thereafter
the reduction ability was maintained for up to 98h in the presence of sucrose,
whereas that in the control declined dramatically to a very low level, within the
same period.
2.3 Importance of Culture Stage

We tried to clarify the nature of the cellular ability of erA to glucosylate


exogenous HQ efficiently, by collecting the cells at various stages of
culture and transferring them to fresh medium. In the experiment shown
in Fig. 7, aliquots of cells were transferred to fresh medium, or, for comparison, to the original medium from which the cells had been removed at
each culture stage. The arbutin-producing ability of cells in fresh medium
kept increasing during the experimental period, while that of the cells
transferred back to the original medium was maximal with 5-day cells. These
results suggest that cells increase in ability to produce arbutin in later

Catharanthus roseus (Periwinkle)

73

Hours ofter the first addition of HQ


Fig. 6. Effect of sucrose on cell viability of Catharanthus roseus during the biotransformation
process. The amount of TTC reduced by the cells was taken as a measure of cell viability. Sucrose
at 6% (e), water (0), and HQ were supplied as described in the legend to Fig. 4. Each value
represents an average of three determinations, with SD. (Yokoyama et al. 1990)

150
,;a
~

....u
~

t:
'Cl

100

\'1

ell

=
=
.t::J.

:0

50

'"

c<l
ell

10

Days
Fig. 7. Change in the ability to produce arbutin during the culture of Catharanthus roseus (CrA).
Cells from early log phase to stationary phase were collected on a 148-mm nylon mesh. Aliquots
of cells (fresh wt.) were suspended in 20ml offresh medium (e) or the original medium (0) from
which the cells had been removed. Every day, 0.2ml of200mM hydroquinone was added until the
cells died. After incubation for the biotransformation, the cell suspensions were homogenized and
arbutin in the supernatant after centrifugation was analyzed by HPLC. Each value represents an
average of three determinations, with SD. (Yokoyama et al. 1996)

stages, but in the original medium they cannot manifest their full ability owing
to an insufficient amount of some component(s) in the medium in the later
stages.
Therefore, the amounts of UDPG were assayed in cells from 3-day and 6day cultures. The control level, before HQ addition, was very high in 3-day

74

M. Yokoyama

6'2-

~ 100
'"
~
0

q
0

50

Ec;:'"
C3
~

Before HQ
addition

16

18

20

22

24

26

16

18

20

22

24

26

150
B

6'2~
~

C3

100

q
0

<.J
;>

50

.....c;:
C)

Bcfore HQ
addition

Hours after addition of 1 mM


hyclroquinone

Fig. SA.B. Change in the UDPG content in cells which had been cultured for 3 (A) or 6 (B) days.
Catharanthus roseus cells were collected after culture for 3 or 6 days and resuspended in fresh
medium by the same method as mentioned in the legend to Fig. 7. HQ was added at 1 mM. UDPG
content in the cells was measured before and at various times after addition of HQ (e). Closed
columns at the left represent original UDPG content in 3-day (A) or 6-day (B) cells before
addition of HQ. UDPG content is represented relative to the original content in 3-day cells, taken
as 100%. (Yokoyama et al. 1996)

cells while in 6-day cells UDPG was hardly detected (Fig. 8), in accordance
with a previous report (Sasamoto and Ashihara 1988). This suggests that
younger cells require more UDPG, presumably for synthesizing the cell wall.
The control level of UDPG does not necessarily reflect the amount which is
available for bioconversion to arbutin. Added HQ may induce new UDPG
formation. Therefore, we assayed the amount of UDPG just after added HQ
(1 mM) had been completely consumed. That amount was expected to reflect
not only the control level, but also the level induced by adding HQ. Figure 8
shows that UDPG was clearly induced in the 6-day cells; the total supplied
amount was larger in 6-day cells than in 3-day cells. In 3-day cells, the supplied
UDPG may be used competitively both for the glucosylation of hydro quinone

Catharanthus roseus (Periwinkle)

75

and for synthesizing cell wall polysaccharides. On the other hand, the activity
for glucosylation of hydro quinone was essentially the same in cells from 3-day,
6-day, and 8-day cultures (data not shown).
The cell volume of erA was estimated to be 11.3mm3 X 10-6 for 3-day
cells and 16.1 mm 3 X 10-6 for 8-day cells. The increase is considered to reflect
the increase in vacuole volume. It is concluded that older cells of strain A
can accumulate arbutin in larger amounts owing to their greater vacuole
volume.
In conclusion, older cells have a greater ability to produce arbutin by
biotransformation because of the larger amount of induced UDPG which is
available for biotransformation and the greater vacuole in which produced
arbutin may be accumulated.
2.4 High-Level Production of Arbutin

In order to achieve high-level production of arbutin, the regulation of


HQ supply is important because HQ is toxic to cultured cells. So the optimum
method of HQ supply was examined using cells in high-density culture
(250-300g fresh wt./1) which had been obtained by appropriate supplementation of medium components in a 5-1 jar fermenter (Inomata et al. 1991).
The effects of intermittent supplies of HQ were first tested; the results are
shown in Fig. 9. HQ at lOmM severely damaged the cells and HQ was not
added further after the first time (Fig. 9A, a). The conversion period of HQ to
arbutin was the longest in the case of HQ supply at 4mM, when the arbutin
yield was 4.3 gil (Fig. 9A, c). The supply at 6 mM gave the best result, the
arbutin yield being 6.0 gil (Fig. 9A, b).
Next the effect of continuous supply of HQ was examined in order to
maintain its concentration in the medium at a selected value. At the outset, in
all experiments HQ was added at 6mM and, after its concentration in the
cultured medium had decreased to 5 mM, 3 mM, or 0 mM, continuous addition
of HQ at 1.4 mmollh was started (equal to the rate at which the cells consumed
HQ). The apparent concentration of HQ in the medium during the continuous
addition was thus maintained at the selected value, i.e., 5mM, 3mm, or OmM.
The effect of this feeding system on arbutin production is shown in Fig. 9B. At
5 mM, the cells died within 1 day and the arbutin yield was very low (Fig. 9B,a).
The yield increased markedly as the apparent HQ concentration in the medium decreased with this feeding system, and was maximal (9.2g/1) at OmM
HQ (Fig. 9B, c). The arbutin content (% of dry wt.) was remarkably high
(45%) and the conversion ratio was 98%. This feeding system minimizes the
contact of toxic HQ with the cells.
In a scaleup test in a 20-1 jar fermenter, the growth rate of cells was similar
to that in the 5-1 jar fermenter. When the cells had grown to a density of 271 g
fresh wt.!1, HQ was added to give a concentration of 6mM, and after this had
been completely consumed, continuous supply of HQ was started at a rate
such that it did not accumulate in the medium. The addition speed of HQ was
4.8mmol/h in this case. The resulting arbutin yields, per suspension volume

76

M. Yokoyama

abc

6g

~10
~

4~
~

.i6

g~4
.c

2 'E
;;l

o~2

x .~

.J-J

B
."

o ~0. 90 a
~ ~ 'E60

e~

<
~

oo'----'---L-J'-'--6-~...........L.....J....---'--~.........

20 1 2340 1 2345

Days after the start of HQ addition

~30

~ ~ - 0r-----+-----------~--------~ 10

1
2

4~

.ie 6

CJ

;;l

~
c

2~

8114
a ~2
~~

OL.....J.....L.....J..--L..---'--'----'--.l.......L-L..L-4---+-4<==L.....LJO

0 2 0 1 234 50 1 234
Days after the start of HQ addition

Fig. 9A.B. Effects of intermittent or continuous supply of HQ on arbutin yield. CrB strain of
Catharanthus roseus was used in these experiments. HQ was added repeatedly (A) or continuously (B) to a jar fermenter as illustrated in the figure, after the cell density had increased to over
250 g fresh weight/I. Glucose was added twice to a concentration of 55 mM, at the starting point of
HQ addition and on the second day thereafter. (Inomata et al. 1991)

(9.1 g/I) and per cell fresh weight (34 mg/g fr. wt,), were essentially the same as
in the 5-1 jar fermenter.

3 Summary
The results of the biotransformation of HQ to arbutin by C. roseus cell culture
may be summarized as follows.
1. Selection of a superior cell strain was important for efficient production of
arbutin. The cell line CrB, with well developed vacuoles, produced much
more arbutin than CrA.
2. Sugars improved the cell viability. The production of arbutin was
greatly enhanced by sucrose or glucose at concentrations of up to 6%, with

Catharanthus raseus (Periwinkle)

77

the enhancement being directly dependent on the concentration of the


sugar.
3. The ability to produce arbutin greatly changed during batch culture. Older
cells possessed higher ability to produce arbutin, but in batch culture they
could not manifest their full ability because of depletion of essential
component(s) in the medium.
4. The mode of HQ supply greatly influenced the yield of arbutin. The yield
increased markedly as the apparent concentration of HQ in the medium
decreased with a continuous feeding system of HQ. The maximal yield was
9.2g/1 when HQ was continuously supplied at a rate such that it did not
accumulate in the medium.

4 Prospects
Arbutin production using C. roseus cell culture has the advantage that
there is no significant competing reaction; the conversion ratio from added
HQ to arbutin was 98%. It may be pointed out that our process is a direct
conversion of HQ and not a de novo synthesis from remote precursors. We
have developed a simple method of purification of arbutin that includes
the use of C l8 columns and recrystallization from organic solvents as the
main steps. The total period of culture is around 18 days; 2 weeks for highdensity cell culture of competent cells, and 3 or 4 days for the biotransformation; this period is relatively short for a plant cell culture process. Pure
arbutin could be produced at a cost of $300-$1000/kg by this method. The cost
range is large because the cost would depend on whether or not
the construction of new facilities for cell culture or for purification of arbutin
is necessary.
On the other hand, glucosylation of HQ by chemical means is not a simple
task; it requires several steps, including blocking the hydroxyl moieties of
glucose via acetylation, conjugation of such OH-protected glucose to HQ, and
saponification for deacetylation. The conjugating reaction requires a high
temperature. The cost is in the same range as that of the biotransformation.
Thus, production of arbutin by biotransformation could substitute for the
chemical process.

5 Protocol
5.1 Cell Culture
Cell suspensions of C. raseus (L.) G. Don cell strain A (CrA) and strain B (CrB), which had been
established at Tohoku University, were subcultured weekly in LS liquid medium (Linsmaier and
Skoog 1965), supplemented with 30g/J of sucrose and 2.2.uM 2,4-dichlorophenoxyacetic acid. Jar
fermenter culture was performed with 5-1 or 20-1 vessels equipped with modified paddle-type
impellers (Tanaka 1982) and spargers of porous sintered metal (40.urn).

78

M. Yokoyama

5.2 Analysis of Arbutin


The cell suspension was homogenized in a Polytron (Kinematica) and centrifuged at 10 000 rpm
for 10 min. Arbutin and HQ in the resultant supernatant were analyzed by HPLC (LC 100
system, Yokogawa Electrics Inc., Tokyo, Japan) on a Capcell Pak CI8 column (Shiseido,
Tokyo, Japan) in 5% methyl alcohol (adjusted to pH 2.5 with phosphoric acid) with monitoring at
230nm.

5.3 Analysis of UDPG


UDPG was analyzed by HPLC (LC 100 system) according to the method of Ashihara et al.
(1987). About I g fresh weight of cells was collected by filtration on a paper filter (No.2,
Advantech, Tokyo, Japan) and transferred to four volumes of cold perchloric acid solution
(6% w/v), followed by disruption with a sonicator (Bioruptor, Cosmo Bio Inc., Tokyo,
Japan). After centrifugation at 18000 rpm for 15 min, the supernatant was neutralized using 20
and 5% KOH solution. The neutralized solution was evaporated in vacuo and the residue was
dissolved in 2ml of phosphate buffer (20mM, pH 7). After standing at 20C for 20min, the
solution was filtered through a paper filter and a guard column (Millipore Inc, USA), then
analyzed by HPLC (LC 100 system) on a Shimpack Wax-I column (4mm X 50mm, 100 A,
Shimadzu Inc., Kyoto, Japan) in 20mM phosphate buffer (pH 7) at I ml/min with monitoring at
260nm.

5.4 Assay of the Enzyme Activity to Glucosylate Hydroquinone


Enzyme assay was performed according to the method of Yokoyama et al. (1990). Crude
enzyme solution was prepared after homogenization of cells in 50 mM phosphate buffer
(pH 5) containing lOmM mercaptoethanol and centrifugation of the homogenate. An aliquot
of the preparation of crude enzyme (lOOml) was added to 80ml of a reaction mixture that
contained lOmM UDP [U- 14C] glucose (0.05mCi), lOmM hydroquinone, 10mM mercaptoethanol and 50 mM phosphate buffer (pH 5), and the mixture was incubated at 37C
for 60 min. The reaction was stopped by addition of 0.2 ml of ethanol containing 0.01 mM cold
arbutin and a 20-ml aliquot of the mixture was developed by TLC. The arbutin fraction detected
under UV light (254nm) was scraped off the plate, and the radioactivity was measured in a
scintillation counter.

5.5 Analysis of Sugars


Sucrose, glucose, and fructose remaining in the medium were analyzed by HPLC on a 5NH2
column (Senshu Inc., Tokyo, Japan) with 80% acetonitrile. Monitoring was done with a refractive
index detector (Erma Inc., Japan).

5.6 TTC Reduction


The ability to reduce triphenyltetrazolium chloride (TTC) was used as a measure of cell viability.
Fresh cells (0.2 g) were washed in 20 ml of phosphate buffer (pH 7) and suspended in 3 ml of a
solution of TTC (0.6% in phosphate buffer, pH7). After incubation overnight in the dark at 26C,
the cells were washed twice with water. Red pigment (reduced TIC) was extracted twice by
heating in 95% ethanol at 85 C for 5 min and quantitated by measurement of the absorbance at
485nm.

Catharanthus roseus (Periwinkle)

79

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Tanaka H (1982) Oxygen transfer in broths of plant cells at high density. Biotechnol Bioeng
24:425-442
Umetani Y, Tanaka S, Tabata M (1982) Glucosylation of extrinsic compounds by various plant
cell cultures. In: Fujiwara A (ed) Proc 5th Int Cong Plant Tissue & Cell Culture. Maruzen,
Tokyo 383 pp
Yamamoto H, Nakagawa K, Fukui H, Tabata M (1986) Cytological changes associated with
alkaloid production in cultured cells of Cop tis japonica and Thalictrum minus. Plant Cell Rep
5:65-68
Yokoyama M (1996) Industrial application of biotransformations using plant cell cultures. In:
Misawa M, DiCosmo F (eds) Plant cell culture secondary metabolism: toward industrial
application. CRC Press, Boca Raton pp 79-121
Yokoyama M, Inomata S, Seto S, Yanagi M (1990) Effects of sugars on the glucosylation of
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31:551-555
Yokoyama M, Inomata S, Yanagi M, Wachi Y (1996) Change of maximal cellular productivity of
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Plant Cell Mol Bioi Lett 13:285-290

VI Centella asiatica (L.) Urban. (Pennywort):


Cell Culture, Production of Terpenoids, and
Biotransformation Capacity
J.M. SOLET, A. SIMON-RAMIASA, L. COSSON, and J.L. GUIGNARD

1 Introduction
1.1 The Plant
Centella asiatica (L.) Urban, synonym C. coriacea Nannfd, previously also
named Hydrocotyle asiatica L. or H. lunata Lam, of the family Apiaceae, has
numerous common names in various languages: pennywort, marsh
pepperwort, Indian waternavelwort (English); asiatisches Wassernabelkraut
(German); b6vilaque, coquelariat, violette marron (French); gotu kola (SriLanka); brahmi, brahmanduki, karivana, mandookaparni, babassa, thankuni,
vellari, vallarai (Indian); talapetraka, anamanitra, korokorona, silabola
(Malagasy); bodila-ba-dinku, tabao en Amhara (African); luo de da, ji xue cao
(Chinese). The large number of native names, especially in India, also shows
its popularity in the Ayurvedic system of medicine (Dandouau 1910; Boiteau
and Chanez 1967).
C. asiatica (Figs. 1,2) is a slender tropical herbaceous plant with crawling
stems, propagating vegetatively by runners (stolons), with entire kidneyshaped leaves (1-3 cm long; 2-4 cm wide) bearing a crenate margin at the tip of
long petioles (5 to 10 times the leaf length). In sunny places, the petioles are
shorter (only 2x the leaf), and petioles and leaves become red due to important anthocyane production. Leaves and short peduncled (2-4-cm) inflorescences arise from a rosette near the ground. The umbels are reduced and
contain mostly two to four white or pink-purple uniform small flowers (2mm)
consisting of five petals, five free stamens, a greatly reduced calyx, an inferior
ovary with two carpels and a stylopodium supporting two styles. The fruit is a
dry, flattened schizocarp with two single-seeded mericarps each with prominent ridges (Boiteau and Ratsimamanga 1956; Chadefaud and Emberger 1960;
Debray et al. 1971; Medicinal Plants of India 1976).
C. asiatica generally grows in damp, shadowed, or swampy areas, but also
in savanna and secondary forest clearings under warm climates of both the
northern and southern hemispheres. It is native to Asia and mainly found in

Laboratoire de Botanique, Faculte de Pharmacie, Universite Paris-Sud, 5, rue 1B Clement, 92296


Chiitenay-Malabry Cedex, France
Biotechnology in Agriculture and Forestry, Vol. 41
Medicinal and Aromatic Plants X (ed. by Y.P.S. Bajaj)
Springer-Verlag Berlin Heidelberg 1998

82

1M. So let et at.

Fig. 1. Centella asiatica (L.) Urban, var. abyssinica in situ

Fig. 2. Centella asiatica (L.) Urban, var. typica

India, Pakistan, and Madagascar; but the plant also grows in tropical and
equatorial Africa, America, and the tropical regions of the New World.
Three varieties are described in relation to geographic origin which are
correlated with some leaf morphology and chemical composition.
- C. asiatica L. var. typica has weakly hairy, typically kidney-shaped leaves

with well-crenulated margins, and is !ound in southern Asia, as far as Madagascar. All references in traditional medicinal concern this variety.

Centella asiatica (L.) Urban. (Pennywort)

83

- C. asiatica L. var. abyssinica shows suborbicular, with softer crenated mar-

gins, quite hairy leaves, and is present in tropical and equatorial Africa.
- C. asiatica L. var. floridana with leaves longer than wide in shape, is found

in America (from southern US to Argentina) and in tropical Oceania.


Each variety can show several chemical compositions: in India, the most
frequent type of the variety typica contains asiaticoside and madecassoside,
whereas a rarer one contains brahmoside and brahminoside (Rastogi and
Dhar 1963).
1.2 Medicinal Importance

The drug is used in both Africa and Asia as a remedy for leprosy and other
skin diseases (Boiteau and Ratsimamanga 1956; Oliver-Bever 1986). Some
tribes use the leaves as a vegetable (in curries or salads). In Chinese traditional
medicine, the plant is used as an antipyretic, detoxicant (blood purifier), and
diuretic. It has also been widely sold as a body strengthener and revitalizer that
can promote longevity. High doses may have sedative or narcotic effects
(Tyler et al. 1988). Triterpenic titrated extract (in French ETCA) has shown
excellent tolerance to short-as well as long-term applications (localized or
administered generally). The drug is also a weak sensitizer (Hausen 1993). In
European pharmacopoeia, indications are cutaneous affections, skin wounds
such as chapping, grazing, insect stings, sun burn, and other light, small burns
(Dalloz-Bourguignon 1975; Poizot and Dumez 1978; Fleurentin 1991), leprous
and other ulcers (Louvet et al. 1976; Babu et al. 1995), and cicatrization after
surgery in gynecology (Basset and Eberst 1980), ophtalmology (cornea lesions), or ORL (eardrum lesions)(Leguay and Greco 1979; Vogel et al. 1990).
It is also indicated in phlebology in the case of veinous diseases (Maggiori
1992). In some recent work, an antiulcer activity of an ethanol extract of C.
asiatica roots has been reported, without establishing any chemical identity
(Sarma et al. 1995).
Several saponins containing the ursane ring system have been isolated
in Centella asiatica. Asiatic acid, made cas sic acid, madasiatic acid, and
asiaticoside are the major terpenoids found in the typica variety. Asiatic acid
(C30H4s0s) is the 2,3,23-trihydroxyurs-12-en-28-oic acid (Fig. 3; Boiteau et al.
1949). Madecassic acid is the 6-hydroxylated asiatic acid (Pinhas et al. 1967).
Asiaticoside (Bontems 1941; Boiteau et al. 1949) is trisaccharide [O-a-Lrhamnopyranosyl (1 ~4) O-fJ- D-glucopyranosyl (1 ~6)] 0-[3-D-glucopyranose
ester of asiatic acid (Polonsky 1949, 1952). Other triterpenes have also
been described, such as madecassoside (the same trisaccharide as in
asiaticoside esterifying madecassic acid), madasiatic acid (a 2,3,6trihydroxylated isomer; Boiteau and Chanez 1967; Pinhas 1969), brahmic acid
(a 2,3,6,23-tetrahydroxy isomer of madecassic acid), brahmoside, brahminoside (respectively a glycoside and an ester glycoside of brahmic acid both
with a rhamnosyl glucosyl arabinosyl-trisaccharide; Rastogi et al. 1960;
Rastogi and Dhar 1963), thankuniside, isothankunic acid, isothankuniside

84

1M. Solet et al.

R1

R2

R3

CH20H

Madecassic acid

OH

CH20H

Madasiatic acid

OH

CH3

CH20H

glc-glc-rhm

CH20H

glc-glc-rhm

Asiatic acid

Asiaticoside
Madecassoside
-~~-~---

--~-,

OH

. ._---------------,._.

--.~

----------

Fig. 3. Main triterpenic compounds of C. asiatica L. var. typica

(a 3,5,6,23-tetrahydroxy isomer esterified with a disaccharide of glucose and


rhamnose; Dutta and Basu 1962, 1968); centic acid (C30H480S)' centellic and
centoic acids (C30H4S06), as well as centelloside (a polysacharide ester of
centellic acid containing 10 glucose and 2 fructose) have also been identified
(Lythgoe and Trippett 1949; Bhattacharyya 1956a,b,c).
It has been shown that C. asiatica enhances collagen synthesis, modulates
growth of fibroblasts, and regenerates skin connective tissue. The pharmaceutical drug (Madecassol Roche Nicholas SA Division SERDEX, previously
named Syntex Laboratories) contains a mixture of asiatic acid, madecassic
acid, and asiaticoside (30/30/40). A positive effect of asiaticoside was already
described in the 1960s (Rosen et al. 1967). Later, Marquart et al. (1990)
showed that collagen synthesis was increased in human foreskin fibroblasts by
a mixture of these three terpenoid compounds: asiatic and madecassic acids
and asiaticoside. More recently, the influence of the three compounds separately on human collagen I was studied (Bonte et al. 1994). The authors
showed a similar rise of collagen I with each compound; they also showed that
the sugar moiety of the molecule did not seem necessary for the biological
activity. The same authors compared asiaticoside and madecassoside (Bonte
et al. 1995). The two triterpenes were shown to stimulate collagen I secretion
by 25-30%, but only made casso side was able to increase collagen III secretion
significantly. The C. asiatica triterpenes, like some mineralocorticoid hormones (therefore also named mineralocorticoid-like substances), in promoting collagen formation, could act as a profibrotic component of wound-healing

Centella asiatica (L.) Urban. (Pennywort)

85

(Weber 1992). Such a role is also proposed for some triterpene derivatives of
licorice root, i.e., glycyrrhetinic acid (Amagaya et al. 1984) and carbenoxolone
(Sloan and Weaver 1968). The pathogenetic mechanism responsible for these
properties and the signal transduction pathway involved in the response remain to be elucidated.

2 In Vitro Approaches
2.1 Tissue and Cell Cultures
Centella asiatica callus and cell suspension cultures were initiated and further
subcultured in Professor Guignard's laboratory (Bister-Miel 1987). To our
knowledge, it is the only cell suspension of this plant still existing. In the past,
some cultures were initiated in Professor Henry's laboratory (Faculty of Pharmacy, Toulouse, France); however they did not provide enough saponin derivatives for industrial application (pers. comm.).
Cell suspensions were obtained after two steps: first, callus cultures initiation on solid medium and then tissue transfer to liquid medium. Seeds were
aseptically germinated and plantlet (stem) fragments were cut into small
Table 1. Centella asiatica callus initiation, callus, and cell suspension growing media

a) Callus initiation medium


Nitsch medium
Sucrose
Agar
Adenine
2,4-D

b) Initial callus-growing medium


MS medium
Glucose
Agar
Adenine
2,4-0

Kinetin

Nitsch and Nitsch (1956)


20gl- 1
7 gl-l
2mgl- 1
0.1 mgl- 1
Murashige and Skoog (1962)
30gl- 1
7 gl-l
1 mgl- 1
0.1 mgl- 1
1mgl- 1

c) Present callus-growing medium


MS medium
Murashige and Skoog (1962)
30g1- 1
Glucose
7grl
Agar
1mgl- 1
Adenine
0.2mgl- 1
2,4-0
0.2mgrl
6-Benzyl-aminopurine
d) Suspension growing medium
MS medium
Sucrose
Casein hydrolysate
2,4-0

6-Benzyl-aminopurine

Murashige and Skoog (1962)


30gl- 1
500mgl- 1
0.2mgl- 1
0.2mgl- 1

86

1M. Solet et al.

~-------I

;;:;

eIJ

.~

:::

....
I....

""

"" "

"

" """ ,," "

InoLululll


"

..c

"

(~/l)

DW Othday)

Dilution at
Week I: lI2
Week 2: lO/66
Week 3-4: 10/41
Week 5-6: 10150
Week 7-11: lI8
Week 12 ->: 1111

"""" "

O+-~-'~--'-~'---~----r---~

10
15
20
Age (weeks)

25

30

Fig. 4. Stabilization of Centella asiatica L. suspension culture


12

DW S7rpm

---0---

10

-eL

:c

,.;:;

eL

....

8
6

,;
~

::::;

10

12

14

16

18

20

Age (days)

Fig. 5. Growth curves of Centella asiatica cell suspension

pieces and placed on modified Nitsch and Nitsch medium (1956) (Table la).
Callus was first subcultured every 4 to 6 weeks on modified MS medium
(Murashige and Skoog 1962) (Table Ib) containing adenine, kinetin, 2,4-D,
and glucose. Insufficient growth led to modification of the 2,4-D concentration
and replacement of kinetin by 6-benzyl-aminopurine (Table lc).
One to 3 months after initiation, when callus had grown enough, it was
transferred to liquid growing medium, where it dissociated spontaneously and
easily, and subcultured on modified MS medium (Table Id). Optimized
growth conditions were rapidly obtained (12 weeks) with appropriate dilution
rate (Fig. 4). The suspension cultures are then maintained by subculturing

Centella asiatica (L.) Urban. (Pennywort)

87

every week at a dilution of 1/1l. The suspensions are placed in 250-ml


Erlenmeyer flasks on an orbital gyratory shaker or in 500-ml to 2-1 flasks on a
roller, both at 180rpm. The temperature is constant at 24 1C and the
culture is submitted to a 16-h photoperiod per day (25.uergm-2s-1 illumination). A typical growth curve (Fig. 5) shows no lag phase, exponential growth
up to day 7, and stationary phase after day 10. The suspension appears a little
chlorophyllous and seems to green up when older; it was used for papaverine
biotransformation (Bister-Miel et aI. 1988), and has also been habituated to a
slower rate of shaking (87 rpm), allowing subculturing only every 14 days (Fig.
5). This suspension growth curve shows a lag phase of 2 days, an exponential
growth up to day 12, and then a short stationary phase up to day 14. This
suspension was used for thiocolchicine biotransformation (Solet 1993), and is
now 14 years old.

2.2 Biotransformation of Papaverine by Centella asiatica Cell


Suspension (Bister-Miel 1987)
After studies of several nonalkaloid-producing suspensions, cells showed
biotransformation of papaverine into papaveraldine, an oxidized derivative (Fig. 6; Bister-Miel 1987). Papaverine is an isoquinoleic alkaloid
[dimetoxy-6,7 (dimetoxy-3',4' benzyl)-l isoquinolein] used as a musculotropic
vasodilator. Papaverine acts as an antagonist of Ca2+ ions inhibiting
cyclic phosphodiesterase (Lugnier et aI. 1972). Accumulation of cyclic AMP
leads to vasodilatation and direct relaxation of smooth muscle (Diamond
1978).
For these experiments, cell suspension culture of Centella asiatica was
subcultured at intervals of 7 days with a dilution of 1111 in a modified MS
medium.

CHjO
------~

"-""-,

/-:--.,

I'

OCH]

Papaverine

CH10/

~ ~ /~
~

'y
)

O/~(~

-"'::i

~;::J~OCH

'~Cfy ~"

~
I

.....

OCH 3

OCH 3

Pa pa veraldine

Fig. 6. Biotransformation of papaverine by cell suspension culture of Centella asiatica L. (BisterMiel1987)

88

1M. So let et al.

100
~

"'e'"'
::
a'"'

80

.......
0

It<

60
40

:c
:::

20

101)

.~

.......

200

400

600

800

Papaverine hydrochloride (mgll)


Fig. 7. Toxicity of papaverine hydrochloride in C. asiatica cell suspension culture. (Bister-Miel
1987)

2.2.1 Toxicity of Papaverine

Concentrations of papaverine (0-1000mg/l) were added to a 7-day-old


cell suspension culture. Cells were collected after a 7-day incubation period
with papaverine by reduced pressure, sieving, and drying at 60C for 48h.
Dry weight (DW) were given as a function of substrate concentration (Fig.
7). A 50% decrease in DW in comparison with the control was observed
for a concentration of 600mg/l. This value allowed biotransformation
experiments.
2.2.2 Papaverine Biotransformation Experiments

Papaverine (200--400mg/l) was added to cell suspension culture at the time of


subculture. After an incubation period of 7 days, cells and medium were
collected, separated, and frozen at -18C. Extraction of metabolites was done
with petroleum ether and acetone. Compounds were identified by TLC and
GC-MS by direct comparison with synthetized standards (Christinaki et al.
1987). Chromatograms revealed the presence of intracellular papaverine (2%
of added substrate). Papaveraldine was detected in both cells and culture
medium. Although no quantification of papaveraldine was performed, the
amount of this product was quite important as compared to the amount of
intracellular papaverine.
Thus, papaverine was converted to its oxidized derivative, papaveraldine,
by a cell suspension culture of C. asiatica. Several other studies have reported
biotransformation of papaverine into papaveraldine by plant cell culture:
Silene alba, Cardamine pratensis, Digitalis purpurea (Bister-Miel et al. 1988),

89

Centella asiatica (L.) Urban. (Pennywort)

Vinca minor (Podvin 1985), Cinchona succirubra, Ptelea trifoliata, Phaseolus


vulgaris, Parthenocissus tricuspidata (Rideau et al. 1988), and Glycyrrhiza
glabra (Dorisse et al. 1988).
2.3 Biotransformation of Thiocolchicine by Centella asiatica Cell
Suspension (Solet 1993)

Cell suspension cultures of C. asiatica of Professor Guignard's laboratory


have also shown capacity of thiocolchicine demethylation and glucosylation
(Fig. 8). Thiocolchicine is a hemisynthetic substrate obtained from natural
colchicine by the replacement of the lO-O-methoxy by a thiomethyl
group (Velluz and Muller 1954). Colchicine and its glucosylated derivative,
colchicoside, are extracted from Colchicum autumnale seeds L. (Petitjean
et al. 1978). Thiocolchicoside (3-0-glucosylthiocolchicine) is a common drug
used as a myorelaxant and an analgesic (Coltramyl, Roussel Laboratories, France). Thiocolchicoside is also chemically obtained from colchicoside by the replacement of the lO-O-methoxy by a thiomethyl group (Bellet
1952).
For these experiments, cell suspension culture was subcultured at intervals
of 14 days with a dilution of 1129 in a modified MS medium.

RIO Rp

4
~

;/'

NHCOCH 3

20--..
I

OCH 3

~8

~
II

10

R1

R2

thiocolchicine

CH3

CH3

2-demethylthiocolchicine

CH3

3-demethylthiocolchicine

CH3

2-0-glucosylthiocolchicine

CH3

glucose

3-0-glucosylthiocolchicine

glucose

CH3

(= thiocolchicoside)
Fig. 8. Structures of thiocolchicine, de methylated products (2- and 3-demethylthiocolchicine),
and glucosylated products (2- and 3-0-glucosylthiocolchicine). (Solet 1993)

90

1M. Solet et al.

2.3.1 Effects of Thiocolchicine on Cell Suspension Culture

Previous studies have shown the antimitotic properties of thiocolchicine


(Jequier et al. 1955). Thiocolchicine as colchicine has a high affinity with
tubulin, inhibiting proliferation of cells (Kang et al. 1990). Inhibition of
cell growth of C. asiatica culture was noted when a low concentration of
thiocolchicine (12 pM, 5 mg/l) was added at the subculturing time. To avoid
this inhibition of cell growth and allow biotransformation experiments,
thiocolchicine was added to a 7-day-old suspension culture (0-850 pM). After
an 8-day incubation period with substrate, cells were separately collected by
reducing pressure, sieving, and drying (DW) at 60C (Fig. 9). A stimulation of
DW was observed with a concentration less than 170pM. Within tested concentrations, no cell division occurred. These concentrations have a strong
antimitotic effect, also attested by a stable number of cells. Microscopic observations revealed great cell distension, with cell diameters varying within a 55160-pm range as compared to the 45-100-pm range found in the absence of
thiocolchicine. Furthermore, we observed a marked increase in the volume of
the vacuole. These observations could give an explanation of the increase in
DW at low thiocolchicine concentration. Concentrations higher than 170 pM
induced a decrease in DW, showing the cytotoxic effect of thiocolchicine. A
50% decrease in DW was observed for a concentration of 800 pM of
thiocolchicine. A concentration of 110 pM of substrate was finally chosen for
biotransformation studies, this concentration allowing a good cell viability
over a long period.
2.3.2 Thiocolchicine Biotransformation Experiments

After a 7-day culture period, thiocolchicine (110pM) was added to a


large-scale suspension culture (3.361) and cultured 12 days more. After
harvesting cells and medium separately, samples were extracted with MeOHH 2 0 (1: 1). After purification and isolation, two pools of metabolites were
identified: 2- and 3-demethylthiocolchicine (TLC, HPLC) and 2-0 and 3-0glucosylthiocolchicine (TLC, HPLC-MS, NMR) by direct comparison with
standards (Fig. 8). Pool size of products were monitored for 12 days after
addition of 110 pM substrate.
Total demethylated products were detected both in the cell extract and in
the culture medium (Fig. 10). They were detected from the 1st day of incubation in the culture medium and from the 2nd day of incubation in cells. After
3 days of incubation, amounts of these products reached stable values both in
cells (2.6pM) and in the culture medium (5.6pM). Demethylated products
were mainly localized in the medium (68%), obviously excreted after
intracellular demethylation activity.
Pool size of glucosylated products in cells and medium was also monitored
for 12 days after addition of substrate (Fig. 11). Intracellular glucosylated
compounds increased gradually during incubation to reach a maximum value
of 10 pM. A very slight amount of glucosides in the medium was detected only

Centella asiatica (L.) Urban. (Pennywort)

91

14
12
10
~

EIOL

::::

Ii

....

.:ji

....

2
0

200

400

600

800

1000

Thiocolchicine (iJ 1\1)


Fig. 9. Toxicity of thiocolchicine in Centella asiatica cell suspension culture. (Solet 1993)

10

::t

'":l
"0

e
Co

"0
OJ

';

...

..c:

'"

0
0

-'

.'i

10 II

12

Days after administration


Fig. 10. Time course of the total de methylated products content in cells (- x-) and culture medium
(-0-) after 11O,LiM thiocolchicine administration. (Solet 1993)

after 6 days of incubation. In contrast with demethylated products, these


results showed that glucosylated products of thiocolchicine were accumulated
only in cells. Traces detected in the medium were probably due to lytic release
of intracellular material.
Considering substrate structure, demethylated products appear to be
the intermediates in thiocolchicine biotransformation which leads to

92

J.M. Solet et al.


10

~
2-

'"

Q,I

<Il

...0

,.-=
'-'

2
I

,./

0
0

10

II

12

Days after administration


Fig.H. Time course of glucoside content in cells (-x-) and culture medium (-0-) after 1l0.uM
thiocolchicine administration. (Solet et al. 1993)

glucosylated products. The direct addition to cell suspension culture of 3-0demethylthiocolchicine confirmed the capacity of these cultures to glucosylate
this intermediate into thiocolchicoside.
Demethylation and glucosylation activity of thiocQlchicine detected in cell
suspension led us to study the enzymes involved. First, crude extract of 14-dayold C. asiatica cells was incubated with thiocolchicine. Formation of 3demethylthiocolchicine (2.5 nmollh/g moist cells) was observed. The boiled
extract did not allow the appearance of metabolite. When NADPH, FAD, and
FMN were added as cofactors to the crude extract, demethylation reached
10nmol/h/g moist cells (8% of added substrate). Neither NADPH alone, nor
FAD and FMN together allowed de methylation. The low activities of all
tested subcellular fractions were probably due to the low abundance of
enzyme in cell suspension. We also noted an increase in microsome bulk
(100000 g pellet) in cells previously incubated with thiocolchicine as compared
to the control. In these subcellular fractions, spectrophotometric measurements showed similar amounts of cytochrome P-450 and P-420 (250-300pmoll
mg microsomal protein). According to subcellular localization and cofactor
requirements, the de methylating enzyme may belong to the cytochrome P-450
monoxigenase family. Research is in progress to identify the oxidizing enzymes which are involved in the oxidative process of papaverine and
thiocolchicine.
Low activity of glucosylation of 3-0-demethylthiocolchicine was detected
in the crude cell suspension extract. Glucosyltransferase was partially purified
by ammonium sulfate fractionation (30-70% salt saturation) followed by gel
filtration on a Sephadex G-lOO column. Active fractions were pooled and
chromatographed on a DEAE Trisacryl column. This protocol resulted in an
increase in the specific activity of the glucosyltransferase by 690-fold as compared with the crude extract (Solet 1993).

Centella asiatica (L.) Urban. (Pennywort)

93

3-0-Glucosylthiocolchicine was formed from 3-demethylthiocolchicine


(3mM) and UDPG (3mM) after incubation with elution fractions (TrisHCl 50mM, pH 7.5, and 2-mercaptoethanol 10mM). The mixture was
incubated at 30C. Glucosyltransferase activity was detected both in the
control cell extract and in the extract of cells previously incubated with
thiocolchicine or 3-demethylthiocolchicine. Research on localization, kinetic
analysis (V m et Km ), and substrate specificity of this glucosyltransferase is in
progress.

3 Summary and Conclusion


Centella asiatica L. is known to contain active triterpenic compounds with an
ursane ring system. Several unpublished studies have demonstrated that cell
suspension cultures of this plant also contain asiatic acid, madecassic acid, and
traces of asiaticoside (pers. comm.). Our suspension culture has shown capacities to biotransform papaverine (with a benzylisoquinolein ring system) and
thiocolchicine (with a tropolone ring system). The enzymatic de methylation
and glucosylation system of thiocolchicine were extracted and the
glucosyltransferase was partially purified.
The biotransformation of papaverine and thiocolchicine by Centella
asiatica cell suspension culture shown in this work are two examples of the
detoxifying of toxic compounds. Indeed, the papaveraldine product is significantly less toxic (1200mg/l) than papaverine (600mg/l) (Bister-Miel 1987).
Thiocolchicoside was also known in animal cells to be less toxic than
thiocolchicine (Jequier et al. 1955). Oxidation of the substrate is the first step
in detoxification, consisting in an increase in molecule polarity. The conjugation of glucose to the hydroxylic group of 3-demethylthiocolchicine leads to a
product which may be accumulated probably in the vacuoles (Kreis and
Rheinhard 1987). In cell suspension culture of C. asiatica, this accumulation
occurs with an increase in cell diameter and especially in vacuole diameter
after addition of thiocolchicine (Solet 1993). Storage of a glucosylated toxic
compound like thiocolchicoside in the vacuoles appears to represent a convenient cellular mode to separate such a compound from its cytoplasmic tubulin
target.
Acknowledgments. This work was supported in part by Roussel-UCLAF Laboratories, whom we
also thank for the generous gift of all authentic samples. The authors are also grateful to Professor
P. Bouchet for providing the photograph of C. asiatica in situ.

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VII Chenopodium album L. (Fat Hen): In Vitro Cell


Culture, and Production of Secondary Metabolites
(Phytosterols and Ecdysteroids)
M.-F. CORIO-COSTET 1, L. CHAPUIS 1, and J.-P. DELBECQUE2

1 Introduction
1.1 Botanical Aspects
The name Chenopodium is derived from the Greek words chenos (goose) and
podos (foot), because the leaves often resemble goose feet. This genus consists
of ca. 120 species, widely distributed over the world, 45 of which have been
described in India.
The family Chenopodiaceae comprises annual plants, with greenish or
reddish leaves, which may be glabrous or hairy, but often floury (Loste 1937).
Several species are known to possess medicinal properties, such as e.
ambrosioides and e. anthelminticum, used as antihelmintic, and which contain
some triterpenoid saponins and flavonol glycosides (Chirva et al. 1971;
Bogacheva et al. 1972; Jain et al. 1990). e. quinoa, growing on Peruvian
tablelands and cultivated by the ancient Incas, also contains flavonol
glycosides, which contribute to its unpleasant taste (De Simone et al. 1990).
However, the seeds are widely used by Andine people in cakes, bread, or
drinks, and are also given to animals, because of their high protein content and
good nutritional value: in the grain crop proteins represent about 20% of the
dry weight (Weber 1978). Several other members of this family have been or
are still used for alimentary purposes: in particular, Beta vulgaris (beetroot)
and Spinacia oleracea (spinach) belong to this group.
e. album, also named fat hen, a common species growing in various parts
of the world, is also used as a leaf vegetable. This plant has received much
attention due to its high protein content in seeds and has been selected for
commercial production (Weber 1978). Moreover, the ability of e. album to fix
nitrogen by association with nitrogen-fixing bacteria in its roots seems interesting (Mukherjee et al. 1985). The plant is medium-sized (Fig. 1) and our
specimens were grown in pots in a greenhouse with a 16-h light period, at 18C
during the night and 25 C during the day.

INRA-Bordeaux, URIv-Phytopharmacie, BP 81, 33883 Ville nave d'Ornon, France


Universite de Bourgogne, CNRS UMR 5548 (Developpement, Communication Chimique), 6
Bd Gabriel, 21000 Dijon, France
I

Biotechnology in Agriculture and Forestry. Vol. 41


Medicinal and Aromatic Plants X (ed. by Y. P.S. Bajaj)
Springer-Verlag Berlin Heidelberg 1998

98

M.-F. Corio-Costet et al.

Fig. 1. Chenopodium album plant grown in the greenhouse

1.2 In Vitro Culture Studies

Because of their nutritional value, the Chenopodium species have been extensively cultured in vitro, in order to select clones and to allow their maintenance
for breeding purposes (Conger 1978). As it appeared interesting to eliminate
the saponins (triterpenic glycosides) responsible for the bitter taste in C.
quinoa seeds, the saponin content of callus and bud cultures has been investigated: sapogenins, such as oleanolic acic, were found in in vitro cultures, as in
roots and in fruits (Burnouf-Radosevich and Paupardin 1983).
To stimulate the production of multiple shoots by in vitro vegetative
propagation, different media have been tested, and the best result was obtained with modified B5 medium (Gamborg et al. 1968), decreasing sucrose,
and increasing nitrate and phosphate salts (Burnouf-Radosevich and
Paupardin 1985).
It has also been possible to establish photo autotrophic and heterotrophic
cell suspension culture (Husemann and Barz 1977; Husemann et al. 1980).
Heterotrophic callus cultures were obtained on MS medium (Murashige and
Skoog 1962) supplemented with 2% sucrose in the dark. In order to establish
green phototrophic cell suspensions, heterotrophic calli were kept on the same
medium under continuous light, and the green mixotrophic calli obtained were
transferred into sugar-free MS medium with CO 2 as carbon source. Such
cultures allowed the comparison of lipid composition: photo autotrophic cell

Chenopodium album L. (Fat Hen)

99

cultures of C. rubrum, such as photosynthetic leaves, contained high amounts


of galactolipids, whereas the heterotrophic cell cultures contained only traces
of these compounds, but were rich in sterols and sterol derivatives (esters,
glycosylesters, etc.).
1.3 Secondary Metabolites: Sterols and Ecdysteroids

In Angiosperms, 24-alkylsterols with 5(6)-unsaturation are generally the major 4-desmethylsterols (Benveniste 1986). However, numerous species have
been reported to deviate from this main line. In particular, Caryophyllalles
(consisting of 12 families with approximatively 10000 species, including
Chenopodiaceae), often synthetize a mixture of L17 -sterols and L1 5-sterols (e.g.,
in Chenopodiaceae; Xu et al. 1990; Salt et al. 1991; Corio-Costet et al. 1993a).
A likely role for such phytosterols is to represent the precursors of particularly
interesting steroids, the ecdysteroids, which have been frequently described in
Caryophyllalles (reviews in Horn and Bergamasco 1985; Lafont and Horn
1989; Camps 1991) and which, in particular, are present in high concentrations
in many species of the Chenopodiaceae family (Toth et al. 1981; Bathory et al.
1982; Dinan et al. 1991; Grebebok and Adler 1991; Grebebok et al. 1991;
Dinan 1992a,b; Corio-Costet et al. 1993a). Initially identified as the molting
hormones of arthropods, ecdysteroids have also been found in numerous plant
species, where they are generally supposed to playa defensive role against
phytophagous invertebrates. Other possible roles for phytoecdysteroids in
plant physiology still remain conjectural (Bergamasco and Horn 1983; Lafont
et al. 1991), but these molecules, which may be relatively abundant in our food,
also have interesting pharmacological effects on mammals (reviews in Simon
and KooIman 1989; Camps 1991).
The in vitro techniques applied to various plant systems producing
ecdysteroids have been considered by several investigators as potential tools
for studying the biosynthesis of these molecules and their possible significance
in plant physiology (e.g., Hikino et al. 1971; McMorris and Voeller 1971;
Ravishankar and Mehta 1979; Camps et al. 1990; Vanek et al. 1990; CorioCostet et al. 1993a,b; Tomas et al. 1993; Svatos and Macek 1994).
We have thus explored in C. album the ability to carry out the production
of such sterols and phytoecdysteroids in vitro, in order to obtain a possible tool
for further studies on the biosynthesis, and the roles of these molecules.

2 In Vitro Production of Sterols and Steroids


2.1 Establishment of Callus and Cell Suspension

The seeds, after appropriate surface sterilization (70% ethanol for 5 min; 5 %
calcium hypochlorite for 20min and, finally, two washes in sterile distilled
water), were sown in glass tubes on a solid medium containing half-strength

M.-F. Corio-Costet et al.

100

MS macro- and micronutrients (Murashige and Skoog 1962) and 0.7% agar,
without hormones, and kept at 25C under continuous light (20.uEm-2s-1).
Hypocotyl segments, leaflet pieces, and shoot apices, taken from seedlings as
explant sources, were placed in Petri dishes containing MS medium with 2,4dichlorophenoxyacetic acid (2,4-D, O.OSmg/l) and kinetin (0.05mg/l) until callus induction. Calli were then transferred into 100-ml Erlenmeyer flasks
containing liquid MS medium (without agar, pH 5.9, with 2,4-D 0.165mg/l and
kinetin 0.107mg/l), incubated on a shaker (ca. 120 rpm) under similar light and
temperature conditions, and subcultured every month. Under these conditions, calli were firm, cream-colored, fast-growing, with a fresh weight increasing from five- to sevenfold at every month of subculture.
Cell suspensions were easily obtained from friable calli, transferred to
Erlenmeyer flasks containing 100mi of the same liquid medium, under similar
light and temperature conditions. Incubations were carried out in an orbital
shaker, and subcultures were made up every month in fresh medium. The cell
suspensions obtained were yellow, fine, and homogeneous: cells became very
round, sometimes forming microcalli. During the first incubation week, the
biomass production was very slow, but increased thereafter more rapidly.
After several subcultures, the biomass maximum was generally obtained in
10 days.
2.2 Ecdysteroids and Sterols in Cell Cultures

Total ecdysteroid and sterol contents observed in C. album, either in vivo or in


vitro, are indicated in Table 1. Ecdysteroid concentrations, in vivo, were
generally higher in plant roots than in leaves. However, cell cultures exhibited
a much reduced production of ecdysteroids, which represented only about 0.629ng/mg dry wt. On the contrary, the average content of sterols was greater in
cell cultures than in plants. It thus appeared that ecdysteroids represented
more than 20% of the total amount of sterols and steroids in roots, but only
10% in leaves and 0.5% in cell cultures. Moreover, the level of ecdysteroids in
cell cultures appeared to decrease with age. Indeed, analyses of the content of
ecdysteroids in the exponential stage of growth of cell culture (7 days old),
or of old cell cultures (20 days old) showed that the young cells contain
9-10 .uglg of ecdysteroids and the old cells contained only 5-6.ug.
Table 1. Sterols and ecdysteroid contents in plants and cell cultures of Chenopodium album. Plants were grown for 5 weeks in
the greenhouse and cell cultures were at the end of exponential
growth phase (1 week after subculture). (Corio-Costet et al.
1993a)
Secondary metabolics
(;ig/g dry wt.)

Leaves

Roots

Cells

Ecdysteroids
Total sterols
Total of sterols and steroids

175
1470
1645

377
1370
1747

10
1980
1990

Chenopodium album L. (Fat Hen)

101

HPLC analysis (Fig. 2A,B) confirmed the presence of 20hydroxyecdysone (20E) as the main ecdysteroid in plants, together with
polypodine B (PoB) and other minor compounds, which is consistent with
previous reports (Toth et al. 1981; Bathory et al. 1982; Dinan 1992a). The cells
containing much lower amounts of ecdysteroids appeared difficult to analyze
by HPLC alone, but enzyme immunoassay (EIA) on HPLC fractions (Fig. 2B)
indicated the presence of several immunoreactive compounds, the two main
ones migrating respectively like PoB and 20E in this NP-HPLC system (11 and
16min retention time).
Analyses of free and esterified sterols from C. album indicated some
differences between whole plant organs and in vitro cultures. Table 2 shows
that, though free sterols were predominant in all cases, the plant contained a
higher quantity of steryl esters than cell cultures (two to three times more).
Interestingly, the highest number of esterified sterols was found in roots (more
than 30%). Although 4-desmethylsterols were the major components in all
cases (more than 55% of total sterols), they were particularly abundant in cell
cultures (more than 95% of total sterols), to the detriment of sterol
biosynthesis intermediates as 4,4-dimethylsterols and 4a-methylsterols (less
than 3 and 2% of total sterol in cells, respectively). These intermediates were
generally more abundant in the esterified fraction, except in roots, where they
represented nearly 30% of the free fraction.
The analysis of 4-desmethylsterols showed the presence of more than 15
different compounds, most of them containing a ~5- or ~7-double bond
(Tables 3, 4). However, the presence of stanols (saturated sterols, ~O) has also
been observed, particularly in roots (more than 4 %). In a gas chromatography
profile of acetate derivatives of a 4-desmethylsterol fraction (Figs. 3, 4), compounds 1,2,5,7,13, and 16 were identified as ~5-sterols and compounds 4, 9,
12, 17, and 18 as ~7-sterols. As indicated in Table 3, ~7-sterols were always
more abundant (>60%) than ~5-sterols 40%), but the extent of these two

Table 2. Distribution and composition of free and esterified


sterols in parent plants and cell cultures of C. album. Plants were
grown for 5 weeks in the greenhouse, and the cell culture was 8
days old. (Corio-Costet et al. 1993a)

Free sterols'
Esterified sterols'
Free sterols
4,4-Dimethylsterols b
4a-Methylsterols b
4-Desmethylsterolsh
Esterified sterols
4,4-Dimethylsterols h
4a-Methylsterols h
4-Desmethylsterolsh

Leaves

Roots

Cells

74.9
25.1

68.6
31.4

88.1
11.9

2.1
2.7
95.2

22.2
7.6
70.2

0.9
1.6
97.5

32.5
12.4
55.1

19.2
6.6
74.2

16.1
3.5
80.4

, Percent of total sterols.


b Relative percentage of total free and esterified sterols.

20E

20
time (min)

10

B
R

2000

1500
1000
500
0
5

10

15

20
time (min)

Fig.2A,B. Chromatogram of a purified extract from Chenopodium album. A UV chromatogram


of ecdysteroids from leaves, showing the two main ecdysteroid peaks identified as polypodine B
(5,20E) and 20-hydroxyecdysone (20E). Conditions: diol column (25 em X 4 mm i.d.) with a
gradient from 5 to 20% ethanol in dichloromethane at 1 ml/min during 20 min. B HPLC of
ecdysteroids from Cells. (A) UV chromatogram (same conditions as A); (B) EIA from the
collected fractions. The results are expressed as nanograms 20-hydroxyecdysone equivalents per
gram dry weight. Before this separation, a blank run was done and measured with the EIA under
the same conditions, in which no residual immunoreactivity was observed. (Corio-Costet et al.
1993a)

103

Chenopodium album L. (Fat Hen)

Table 3. Analysis of the 4-desmethylsterol fraction. Percentage of


different unsaturated classes of 4-desmethylsterols. t..5-sterols
were unsaturated at C, (2, 5, 7. 13, 16) t..O-sterols were saturated
compounds (3. 6, 8, 14); t..7-sterols were unsaturated compounds
at C7_N (4, 9, 12, 17, 18), t..5,7-sterols were double unsaturated
compounds at C7 _, and C5-1> (10.15). (Corio-Costet et al. 1993a)
4-Desmethylsterols

Leaves

Roots

Cells

t..5-Sterols"
Ml-Sterols
t.. 7 -Sterols
t..5,7-Sterols
t.. 7I t..5

36.3
1.2
61.5
1.0
1.7

Percentage
30.3
4.2
65.0
0.5
2.2

15.1
1.8
81.5
1.6
5.5

Free sterols
t..5-Sterols
t..7-Sterols
t.. 71 t..5

42.5
56.1
1.3

32.4
62.7
1.9

14.7
82.3
5.5

Esterified sterols
t..5-Sterols
t..7-Sterols
t..71t..5

17.7
77.3
4.6

19.2
75.6
4

17.4
76.6
5

" Percentage of total sterol.

Table 4. Composition of 4-desmethylsterols in the free and esterified fraction isolated from plant
and cell cultures of C. album. F, free sterol fraction; ES, esterified sterol fraction
4-Desmethylsterols

[1]
[2]
[3]
[4]
[5]
[6]
[7]
[8]
[9]
[10]
[11]

[12]
[13]
[14]
[15]
[16]
[17]
[18]

Cholesterol
Cholestanol
Lathosterol
Campesterol
Campestanol
Stigmasterol
Stigmastenol
t.. 7-Campestenol
t..5,7-Stigmastenol

**

Spinasterol
Sitosterol
Stigmastanol
t..5,7 Sitostenol
Isofucosterol
t.. 7-Stigmastenol
t..7 -A venasterol

mg of total desmethylsterols
Free form
Esterified form

RRT

1.12
1.17
1.18
1.23
1.30
1.32
1.35
1.36
1.37
1.40
1.41
1.42
1.43
1.44
1.46
1.47
1.51
1.52

Leaves (%)

Roots (%)

Cells (%)

ES

0.7
2.0
0.2
1.6
1.5
0.1
8.7
0.5
3.4
0.4
0.2
15.8
24.6

0.5
4.0
0.3
5.5

0.2
1.6

1.1

2.3
0.3
10.0
1.0
2.5
0.1
0.5
27.2
16.9
3.0

1.0
2.3
1.0
8.0
9.1
1.5
2.2
2.0
51.0
10.5

5.0
34.9
0.4
1.25
1.05
0.19

ES
0.6
3.0
0.1
0.4
1.1
0.5
1.8
0.8
4.5
0.3
0.4
14.5
9.0
2.2
0.9
3.7
45.7
10.5

1.4
31.3
1.7
0.98
0.66
0.32

ES

0.1
0.5

0.2
3.0
0.1

0.4

0.5

4.0
0.2
2.7

3.2
2.5
0.2
0.3

0.3
33.1
7.0
1.2
1.4
2.6
45.6
0.9

25.1
7.2
1.0
2.1
3.3
49.0
2.1
l.89
1.69
0.20

Unidentified compound, but seems to be desmosterol isomer with characteristic ions at m/e: 366
(M+-OAc).
** Unidentified compound.
, Relative percentage of total free or esterified sterols.
h Undetectable.

104

M.-F. Corio-Costet et al.


17

12

18
13

CHOL

16

14

Fig. 3. Gas chromatography profile of acetate derivatives of 4-desmethylsterols from C. album


roots. CHOL Cholesterol added as internal standard, elution time was 24.53 min. Other peaks
were steryl acetates of: 1 unidentified compound, isomer of desmosterol; 2 cholesterol; 4
lathosterol; 5 campesterol; 6 campestanol; 7 stigmasterol; 8 stigmastenol; 9 ~7-campestenol; 12
spinasterol; 13 sitosterol; 14 stigmastanol; 15 ~5-7-sitostenol; 16 isofucosterol; 17 ~7- stigmastenol;
18 ~7-avenasterol. GC conditions: OV1 capillary column (30m X 0.32mm i.d.), carrier gas H 2,
temperature program rising from 60 to 240C (10 C/min) and 240 to 300C (2C/min). Sterol
identification was determined by GC-MS

categories is noticeable because only a few plant species have been reported to
have such a peculiarity: indeed, plants rich in ,17-sterols are generally reported
to contain almost exclusively such compounds (Xu et al. 1990).
Interestingly, ,17-sterols were found to be much more abundant in cell
cultures (more than 80%) than in plants, where the ,17/,15 ratio was not very
different in leaves and in roots. Considering free and esterified fractions, the
level of ,17-sterols was almost similar in the two fractions of cell cultures,

Chenopodium album L. (Fat Hen)

105

HO

Fig.4. Structure of 4-desmethylsterols found in C. album. 1 ~()-sterols or stanols; 2 ~5-sterols; 3 ~7sterols; 4 ~5.7-sterols; R structure of the side chain. [1] isomer of desmosterol (1A); [2] cholesterol
(cholest-5-en-3/3-01) (2B); [3] cholestanol (cholestan-3/3-01) (J B); [4]lathosterol (cholest-7-en-3/301) (3B); [5] campesterol [(24R)-24-methylcholest-5-cn-3j3-01] (2C); [6] campestanol [(24R)-24methylcholestan-3J3-01] (1 C); [7] stigmasterol [(22E)-(24S)-24-ethylcholesta-5,22-dien-3/3-ol]
(2E); [8] stigmastenol (24-ethylcholestan-22-en-3J3-ol) (1 E); [9] ~7-campestenol (24methylcholest-7-en-3/3-01) (3C); [10] ~5,7-stigmastenol (24-ethylcholesta-5,7,22-trien-3/3-01) (4E);
[12] spinasterol (24-ethylcholesta-7,22-dien-3/3-01) (3E); [13] sitosterol [(24R)-24-ethylcholest-5en-3j3-ol] (2D); [14] stigmastanol (24-ethylcholestan-3j3-0J) (1 D); [15] ~5,7-sitostenol, 24ethylcholesta-5,7 -dien-3/3-ol (4D); [16] isofucosterol [(24Z)24-ethylcholesta-5,24(24 1)-dien-3J3-01]
(2F); [17] ~7-stigmastenol, dihydrospinasterol (24-ethylcholest-7-en-3J3-ol) (3D); [18] ~7avenasterol, 24-ethylcholesta-7.24(24 1)-dien-3/3-01 (3F)

M. -F. Corio-Costet et a\.

106

whereas it was in favor of the esterified fraction in leaves and roots, which
indicated a preferential esterification of .-17-sterols in intact plants, but not in
cells in vitro.
From a qualitative viewpoint, major .-17-sterols were .-17-stigmastenol (17,
see Table 4) and spinasterol (12). Sitosterol (13) and stigmasterol (7) were the
major .-15-sterol, whereas cholesterol (2), lathosterol (4), stanols (3, 6, 8, 14)
and .-15,7-sterols (10, 15) were minor compounds. The distribution of sterols in
free or esterified fractions showed some differences between cells in vitro and
plants, in particular of free sterols, for which higher levels of .-17 -sterols (12, 17)
and lower quantities of stigmasterol (7) or sitosterol (13) were found in cells
compared to plants.
2.3 In Vitro Incorporation of Radiolabeled Mevalonate
Sterol and steroid biosynthesis were investigated in isolated cells of
Chenopodium album after incubation of radiolabeled 14C mevalonic acid
(5.uCi/flask) for up to 7 days and analyzed as previously described (Delbecque
et al. 1995). After cell extraction, a significant incorporation of radioactivity
was obtained into sterols, more than 12% of the incorporated mevalonate
after 7 days of culture, as shown by radio-TLC analysis (Fig. 5, Table 5).

Table 5. Radioactivity recovered after 3 and 7 days of incubation of DL-[2-14C] mevalonic acid
sodium salt (5,uCilfiask; AS: 49.4mCi/mmol) in sterol and ecdysteroid fraction. L. (Chapuis and
M.-F. Corio-Costet, unpub\.)
Radioactivity recovered (%)
3 days'

7 days'

Incorporated in cells
Incorporated in medium

57.5
42.5

59.4
40.6

Incorporated in sterol fraction


Free sterols
4-Desmethylsterols
4a-Methylsterols
4,4-Dimethylsterols

l1.1 b
97.8'
0.8'
1.4'

10.9b
92.8'
3.2'
3.9'

Esterified sterols
4-Desmethylsterols
4a-Methylsterols
4,4-Dimethylsterols

1.78b
59'
6.2'
34.8'

1.3 b
66.3'
6.1'
27.6'

Radiactivity recovered post-Sep-Pak separation


SPO
SP20
SP60
SP80
SP100

86.1'
79.4'
4.2'
5.9'
3.8'
6.7'

97.7'
76.9'
4.9'
8.5'
5.5'
4.2c

, After 3 and 7 days of incorporation 9.7 to 11.8 X 106 cpm.


b Percentage of radioactivity incorporated in cells.
, Relative percentage to the sterol fraction or ecdysteroid fraction.

Chenopodium album L. (Fat Hen)

107

'"

,.,
'"

:a

;;;

::i'

1?\

0>

8
....

:x
'"'

s
'"

::i
0

E~

'"
~

:~
~

Fig. 5. Radio-TLC-analysis of purified extract from C. album cells radio labeled with mevalonic
acid after 7 days. Extract was chromatographied on silica plates (0.2 mm) with dichloromethane as
eluting solvent. A Baseline; B 4-desmethylsterol fraction; C 4a-methylsterol fraction; D 4,4dimethyl sterol fraction; E esterified sterol fraction. (M.-F. Corio-Costet and L. Chapuis, unpub!.)

Moreover, after purification of ecdysteroids by C-18 Sep-Pak extraction, the


SP60 fraction (elution by 60% methanol) was analyzed in RP-HPLC, which
showed a significant incorporation of radioactivity in a peak having the same
retention time as authentic 20E (Fig. 6). Since, in fact, 20E and PoB cannot be
separated in this RP-HPLC system, this labeled peak most likely corresponded to the sum of these two endogenous ecdysteroids. This peak was
significant, as it approximately represented 0.2 to 0.4 % of the total radioactivity, which is consistent with the concentration of ecdysteroids vs. total sterols
(ca. 0.5%, see Sect. 2.3) in cell cultures.

3 Summary and Conclusion


Cell cultures have been obtained in vitro from Chenopodium album, a plant
producing ecdysteroids. In order to better understand the mechanisms of
biosynthesis and function of free and esterified sterols and their relationships
with ecdysteroids, sterol and ecdysteroid contents have been compared in
plant and cell cultures. Free and esterified sterols were isolated and identified.
Sterols such as cholesterol and t,s.7-sterols were detected, which may be the

'"
E

.~

I-

108

M.-F. Corio-Costet et al.


(/)

:::l

o
U

20E
1000

~~--------------~----------------~r-------~~

10

20

time

Fig. 6. Radiochromatogram following RP-HPLC separation of a purified extract (SP60 fraction)


of Chenopodium album cells in vitro, after incorporation of radioactive [14C]-mevalonic acid for 7
days. Abscissa indicate the time in min; ordinate the radioactive counts (6s update time) measured
by a Flow-One apparatus. The horizontal bar indicates the retention time of standard 20hydroxyecdysone (20E). HPLC conditions: Merck Lichrospher RP-18 column (125 x 4mm); 12 to
38% acetonitrile in water at 1 mUmin for 20 min

precursors of ecdysteroid biosynthesis. Ecdysteroid contents were much lower


in quantity in cell cultures than in plants, confirming, however, the presence of
20E. Moreover, the incorporation of radiolabeled mevalonic acid into 20E
suggested that cell cultures of C. album remained able to synthesize
ecdysteroids, but at a reduced rate.
Thus, it could be interesting to use such isolated cells as a model for
studying the potential induction of ecdysteroid biosynthesis in plants. Indeed,
cell cultures undoubtedly still have the genetic capacity to produce such compounds, and it may be interesting to reactivate this capacity by using different
treatments such as modifications of hormonal balance or of various nutrients
(level of sucrose, azote source, etc.), such as additions of activators or inhibitors of Cyt-P-450 enzymes or such as the effects of stresses or elicitation.
Moreover, isolated cells of C. album present a great interest because they
have kept the capacity to produce high concentrations of sterols, contrarily to
other models (e.g., isolated cells of Serratula tinctoria; Corio-Costet et al. 1996),
and that the sterol accumulation in these cells undoubtedly reflects the existence of a late block between sterol and ecdysteroid biosyntheses. Indeed, the
occurrence of cholesterol and i17-sterols is interesting in relation to the biogenesis of ecdysteroids (Sauer et al. 1968; Davies et al. 1980; Adler and Grebebok
1995). Cholesterol is a precursor of ecdysteroids in insects and L17-cholesterol
is generally considered to be the first intermediate in the transformation of

Chenopodium album L. (Fat Hen)

109

cholesterol into ecdysteroids in these animals (Rees 1985). The occurrence of


low but significant quantities of !':J.5,7 alkyl sterols in C. album suggests that
these compounds could be storage forms of biosynthetic intermediates of
ecdysteroids. In plants, the occurrence of !':J.5-sterols among 4-desmethylsterols
in species producing predominantly !':J.7-sterols is considered as rare (Xu et al.
Squalene

(21 %) 4,4-DIMETHYLSTEROLS

STEROLS
78.3%

(7%) 4a-METHYLSTEROLS

(72%) 4-DESMETHYLSTEROLS

t.5-sterols (30%)
t.7-sterols (65 % )
t.5-7-sterols(1%) ~

stanols(4%)

HO~------'
Lathosterol (0.13%)

""

""

"

HO

7-Dehydrocholesterol (trace)

~~ /
Cholesterol (2%)

~------------~"

tOROR

ECDYSTEROIDES
21.7%

~
''''''''

HO

OR

:.

UII

JlO

ID

20-Hydroxyecdysone

OR

-~

oo~

Polypodine B

Fig. 7. Proposed scheme of sterol biosynthesis for ccdysteroid production in Chenopodium album. The various percentages are the percentage of total sterols of three sterol classes (underlined) and of the most important sterols. (M.-F. Corio-Coster, unpubl.)

110

M.-F. Corio-Costet et al.

1990). L\7-Sterols are, indeed, frequently interpreted as biosynthethic intermediates during the isomerization of ,18- to L\5-sterols. It has therefore been
proposed that high levels of L\7(8)-sterols result from a genetic block or from
a defective control of the enzyme catalyzing 5(6)-sterol synthesis (Nes 1977;
Caputo et al. 1983). This hypothesis is reinforced by a recent work on
Arabidopsis mutants, deficient in sterol biosynthesis, which accumulate ,17and L\5-sterols (Gachotte et al. 1995). The unusual ratio of L\7/L\5-sterols in C.
album plants and cells may thus reflect an intermediate regulation of the kinetic
steps, such as the ,15 -desaturase, an enzyme of the sterol biosynthesis.
Thus, from our results on C. album, a scheme of the flux from sterol to
ecdysteroid biosynthesis (Fig. 7) is proposed, where the presence of cholesterol derivatives such as ,17 (lathosterol) and ,15-7 derivatives most likely plays
an important role in ecdysteroid biosynthesis. A careful examination of the
kinetics of these molecules in plant and cell cultures at various stages may
undoubtedly improve our understanding of the biogenesis of ecdysteroids in
C. album in the future.

4 Protocols
4.1 Isolation and Analysis of Ecdysteroids
Plant material was extracted as described previously (Corio-Costet et al. 1993a). The eluted
fractions of ecdysteroids were evaporated and submitted to HPLC (high performance liquid
chromatography in reverse or normal phases, i.e., RP or NP-HPLC) and/or enzyme immunoassay
(EIA), as described by Corio-Costet et al. (1993b).

4.2 Isolation and Analysis of Sterols


The sterol composition of plant organs and cell cultures was determined as previously described
(Benveniste et al. 1984). Sterol profiles were determined from 1 g dry weight samples. Plant tissues
were harvested after 5 weeks of growth (1 week for cell culture), frozen, and lyophylized. The
various sterol fractions were acetylated at room temperature overnight as previously described
(Costet et al. 1987; Co stet-Corio and Benveniste 1988) and identified by capillary gas-liquid
chromatography and gas-liquid chromatography-mass spectrometry.
Acknowledgments. The authors wish to thank N. Pitoizet for technical assistance, and C. Malosse
for his assistance in monitoring the GC-MS. This work was partly supported by the Conseil
Regional de Bourgogne.

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VIII Comus kousa (Dogwood): In Vitro Culture,


and the Production of Tannins and Other
Phenolic Compounds

1 Introduction
The genus Comus (family Cornaceae) consists of about 40 species, nearly
all of which are native to the northern hemisphere. The name dogwood is
a corruption of dagwood or dagger wood because the daggers (for skewering meat) were made from the wood of some sorts (Everett 1981; another
tradition also exists that the extract of the barks was used for the treatment
of skin disease in dogs). Comus plants, with flower clusters surrounded
by large, spreading, petal-like bracts, have great decorative merit for
garden and landscape trees. C. florida (eastern flowering dogwood) and C.
nuttallii (western flowering dogwood) are especially popular trees in North
America.
Comus kousa of Japan and Korea is also often used as a garden tree; the
clusters of flowers (Fig. 1) and the brilliant red leaves in autumn look attractive
under city conditions. The varieties C. kousa chinensis (from China), which is
not botanically very distinct from the typical species, and C. kousa Milky Way,
which blooms unusually freely, occur in Japan. C. capitata, whose four bracts
turn yellow, often decorate the mountainside (evergreen forests) in the
Himalayas.
For medicinal use, C. officinalis Sieb. et Zucc., native to China, is often
employed. The extract of the dried fruits, containing organic acids (malic
acid, tartalic acid, gallic acid, etc.) and fatty oils (palmitic acid, oleic acid,
etc.) is a popular tonic, an astringent, and a hemostatic in East Asian
countries (Mitsuhashi 1988). The fruits (comus fruits) have been often one of
the ingredients in traditional prescriptions for preventing and improving
symptoms of aging, including pollakiuria and cataract (Yazaki and Okuda
1993).
Some other dogwood species provide food for wildlife (Eyde 1988); for
example, the fruits of C. mas L. can be used to make jelly. The bark of some

I Department of Applied Biological Sciences, Faculty of Agriculture, Saga University, 1 Honjo,


Saga 840, Japan
2 Chichibu Onoda Cement Corporation, Tsukuba Biotechnology R&D Center, 25-13, l-Chome
Kannondai, Tsukuba, Ibaraki 305, Japan
3 Tsukuba Medicinal Plant Research Station, National Institute of Health Sciences, 1
Hachimandai, Tsukuba, Ibaraki 305, Japan

Biotechnology in Agriculture and Forestry, Vol. 41


Medicinal and Aromatic Plants X (ed. by Y.P.S. Bajaj)
Springer-Verlag Berlin Heidelberg 1998

114

K. Ishimaru et al.

Fig. 1. Plant of Comus kousa grown at Saga University. (Photograph Ishimaru, July, 1995)

species also yields a compound that can be substituted for quinine (Brinkman
1974).
Comus plants are also rich in tannins, which can be classified into two
large groups, i.e., hydrolyzable and condensed tannins. Some chemical investigations, particularly on this medicinal plant (Okuda et al. 1981, 1984; Hatano
et al. 1989a,b; Lee et al. 1989) have clarified the major polyphenol constituents
of the plant to be hydrolyzable tannins, the metabolites of gallic acid.

2 Tannin Constituents in Comus Plants


The tannin contents in the leaves of eight cornaceous plants (c. kousa
chinensis, C. kousa Milky Way, C. kousa Gold Star, C. kousa Satomi, C. kousa
Snowboy, C. capitata Mountain Moon, C. drummodii Eddie's White Wonder,
and C. officinalis), collected in November in Japan, were determined. The
tannins investigated are shown in Fig. 2; i.e., gallic acid (1) and related
galloylglucoses, [3-glucogallin (2) (Kashiwada et al. 1984), 1,2,6-tri-O-galloylf3-D-glucose (3) (Haddock et al. 1982a,b), 1,3,6-tri-O-galloyl-f3-D-glucose (4)
(Haddock et al. 1982a,b), 1,2,3,6-tetra-O-galloyl-[3-D-glucose (5) (Haddock et
al. 1982a,b), and 1,2,3,4,6-penta-O-galloyl-f3-D-glucose (6) (Haddock et al.
1982a,b), flavan 3-01s (+ )-catechin (7) and (+ )-gallocatechin (8), and condensed tannin procyanidin B-3 (9) (Thompson et al. 1972). These compounds
were analyzed by HPLC; column; TSK-gel ODS 80Ts (4.6mm i.d. X 250mm),
mobile phase; MeCN-1 mM tetrabutylammonium (pH 2.9 by CH3COOH)
(1:9~4:1, in 30min), flow rate; O.6ml/min, column temperature; 40C, R t

Comus kousa (Dogwood)

115
OH

OH

.OC-Q-OH

G:

HOOC-Q-OH
OH

OH

p-glucogallin (2) : R'_4=H

1,2,6-tr~-O-galloyl-p-D-glucose (3) : R" 4=G, R2,3=H


1,3,6-tn-O-galloyl-6-D-glucose (4): R2 4=G, R" 3=H
1,2,3,6-tetra-O-galloyl-p-D-glucose (5): R" 2, 4=G, R3=H
1,2,3,4,6-penta-O-galloyl-p-D-glucose (6): R'_4=G

gallic acid (1)

OC

:;;-.
HOW":::'"

:::".1

HO

1 OH

OH

OH

OH

6c

HOW":::'"
:;;-.

(+ )-catechin (7)

:::".1

HO

0H

1 OH

OH

(+)-gallocatechin (8)

OC
OC
OH

HOW":::'"
:;;-.
1 OH
:::".1

OH

HO:
HOycx=
0 ..,:::".
:;;-:::".1
HO

OH

1 OH

OH

procyanidin B-3 (9)

Fig. 2. Phenolics of cornaceous plants

(min): 2 (6.3), 1 (8.5), 8 (11.2), 9 (16.1), 7 (17.1), 3 (19.2), 4 (20.5), 5 (22.8) and
6 (23.8).
In all plants, the major tannin was 6 (0.13~ 1.46% as dry weight; Fig. 3). In
C. capitata Mountain Moon the content of 7 was characteristically high
(0.36%, as dry wt.).

3 In Vitro Approaches
Yazaki and Okuda (1989, 1993) raised callus and cell suspension cultures of C.
officina lis and studied their tannin production. The callus and suspension
cultures, in LS medium (Linsmaier and Skoog 1965) with auxins [2,4dichlorophenoxyacetic acid (2,4-D), naphthalene acetic acid (NAA), or
indole-3-acetic acid (IAA)] and cytokinins [benzyladenine (BA) or kinetin],
produced much higher contents of trio, tetra- and penta-galloylglucoses
compared to those of the intact plant. C. stolonifera callus has been used
for physiological studies (Niki et al. 1978, 1979; Yoshida and Tagawa 1979).
Successful micropropagation by shoot cultures from apical buds of C.
florida (Coker 1982) was achieved showing nondormant bud proliferation
to be best on Knop's medium supplemented with either 1 or 2mg/l BA. In
an in vitro proliferation system for Cornus species, Trigiano et al. (1989,
1992) also studied somatic embryogenesis from immature zygotic embryos of
C. florida, and indirectly from embryogenic calli derived from zygotic em-

K. Ishimaru et al.

116
1.6

...

_ IA
~

1.6

...

Comus kousa var. chinensis

'0; 1.2

'0; 1.2

:t

~ 1.0

C' 1.0

a 0.8

"CI

; o.~
t\!

06

OA

0. 2

0.0
2
1.6

...

1.4

OIl

'w 1.2

0.6

OA

0. 2

0.0

6
1.6

Comus kousa 'Satomi'

t '1.0

~ 1.0

"CI

; 0.8

t\!

t\!

0.6

U.8

0.6

0.4

OA

0. 2

0.2

,=..,

0.0
2

~
:t

'w l.2

0.0
6

Cornus kousa 'Snowboy '

~
&~

~ 1.0

1.6
1.4
1.2

F
Cornus capitata
'Mountain Moon '

~ 1.0

"CI

"CI

; 0.8

~ UJ~

t\!

'":t 1.2

:t

_ 1.4

1.4

Comus kousa 'Gold Star'

"CI

1.6

Cornus kousa 'Milky Way '

:t

"CI

t\!

_ 1.4

S:?

0.6

0.6

0.4

0 .4

0.2

0. 2

0.0
9

0.0
4

Fig. 3. Polyphenol contents in Comus plants. (K. Ishimaru et aI., unpuhl.)

bryos. Here, some in vitro approaches for Comus plants, such as


micropropagation via shoot cultures of C. kousa chinensis, C. kousa Milky
Way, C. capitata Mountain Moon, and callus and cell suspension cultures of C.
kousa are described, The tannin productions in these tissue cultures, at the
same time comparing the profiles with those of the intact plants, are also
investigated, The work on micropropagation (Trigiano et al. 1992) and secondary metabolites (Yazaki and Okuda 1993) in various species of Comus has
been reviewed.

Comus kousa (Dogwood)

..

1.6

_ 1.4

.=

'"
1.2
~

117

1.6

Comus drummodi;
'Eddie 's White Wonder'

i:' 1.0
."

-:: 1.4

c;,

H
Cornus officinaJis

'"
1.2
~

i:' 1.0

."

~ 0.8

~ 08
~

0.6

0.6

0.4

0. 4

0.2

0.2
0.0 ..I-_ _ _....r::~='_'_..L_ _ __
4

1 : gallic acid, 2: p-glucogallin, 3: 1, 2, 6-tri-O-galloyl-p-D-glucose,


4: 1, 3, 6-tri-O-galloyl-p-D-g1ucose,
5: 1,2, 3, 6-tetra-O-galloyl-p-D-glucose, 6: 1, 2, 3 ,4, 6-penta-O-galloyl-p-D-glucose,
7: (+)-catechin, 8: (+)-gallocatechin, 9: procyanidin B-3
Fig. 3. Continued

3.1 Shoot Cultures of Cornus Plants


3.1.1 Establishment of Cultures
Multiplication of Comus plants by axillary shoot proliferation is currently
the most commercially valuable method for micropropagating selected
cultivars. Young branches of C. kousa chinensis, C. kousa Milky Way, and
C. capitata Mountain Moon were collected and sterilized by usual methods.
The axillary buds and shoot apices were cut off and placed on BW medium
(Sato 1991) under illumination. Successful proliferation of shoot tissues was
obtained (Fig. 4, showing only C. kousa chinensis). The proliferated shoots
fill the culture bottle (450cm 3, containing 90ml medium) in 2-3 months of
culture.
Active and vigorous rooting in in vitro plants is important for obtaining
healthy plantlets which will be harvested and successfully acclimatized for field
planting. In vitro root formation of these plants was obtained on BW medium
containing NAA, indole-3-butyric acid (IBA), and active charcoal (Fig. 5,
showing only C. kousa chinensis). Within 1-2 months of culture, (100%)
rooting occurred.
The proliferated shoots with well-developed roots, after acclimatization
by the usual method (gradually reducing humidity and increasing light intensity), were successfully transplanted to pots (Fig. 6, showing only C. kousa
chinensis). Thus, in vitro propagation techniques are very useful for the rapid
mass production of selected clonal plants of Comus.

118

K. Ishimaru et al.

Fig. 4. Shoot cultures of Comus kousa


chinensis cultured for 8 weeks in BW medium in the light at 25C. (Photograph
Kamiya, September, 1995)

Fig. 5. Root proliferation of Comus kousa


chinensis cultured for 8 weeks in BW medium containing NAA, lEA, and active
charcoal in the light at 25C. (Photograph
Kamiya, September, 1995)

3.1.2 Tannin Contents in Shoot Cultures

Tannin (1-9) contents in shoot cultures of C. kousa chinensis, C. kousa Milky


Way, and C. capitata Mountain Moon are shown in Figs. 7-9. In all species,
high contents of tannins were observed in leaves, and those in stem and root
were fairly poor. Although the major tannin in the intact plants was 6

Comus kousa (Dogwood)

119

Fig. 6. Potted plant of Comus kousa


chinensis. (Photograph Kamiya, September,
1995)

(Fig. 3A,B,F), the main component observed in in vitro plants was lower
molecular weight phenolic 2 (1.38-2.70%, as dry wt.). These observations
indicated that tannin metabolism (especially the gallic acid metabolism) in
these plants might vary with aging, seasonal change, and culture conditions.
3.2 Callus and Cell Suspension Cultures of C. kousa

3.2.1 Establishment of Callus Cultures

Calli of C. kousa were derived from leaf segments of the parent plant. For the
induction of the calli, ten types of MS solid media (Murashige and Skoog 1962)
supplemented with various combinations of 2,4-D, NAA, IAA, and BA
(Table 1) were used. The addition of 0.1 mg/l BA to the medium evidently
promoted callus formation. Particularly, on the medium with NAA (0.5 or
2mg/l) the existence of BA was essential for callus induction. Among ten
media, that containing 0.1 mg/12,4-D and 0.1 mg/l BA (medium A) was best for
the induction of the callus.
3.2.2 Isolation of Tannins

From the calli cultured on medium A for 5 weeks in the dark, four phenolic
compounds 2 and 7-9 were isolated. Among them, 2, a monogalloyl ester of

0.0

0.4

0.8

1.2

1.6

2.0

Ii

='

0.0 1

0.4

0.8

1.2

'--3
4

=:==='
~

1\

'-=='
7

I""';;:::'"

If

-=
'"eo

-=
'"eo

to

1.6

stem

to

:r;

'0;

2.0

0.0

0.4

0.8

1.2

1.6

2.0

'i

Ii

root

1 : gallic acid, 2: fl-g1ucogallin, 3: 1,2, 6-tri-O-galloyl-fl-D-glucose, 4: 1,3, 6-tri-O-galloyl-fl-D-glucose,


5: 1,2,3, 6-tetra-O-galloyl-fl-D-glucose, 6: 1,2,3,4, 6-penta-O-galloyl-fl-D-glucose, 7: (+)-catechin,
8: (+)-gallocatechin, 9: procyanidin B-3

leaf

,;:

Fig. 7. Polyphenol contents in in vitro plant of Comus kousa var. chinensis. (K. Ishimaru et aI., unpubL)

If

'"eo

-=

to

:r;

'0;

,;:

P"

0>

rg..

......

..tv

2.8

2.0

5 Ii
7

R
9

0.0

0.0
3

0.4
2

0.8

1.2

1.6

2.0

2.4

2.8

0.4

.
..'"
ts'?

.;:;

til)

:c

0.8

1.2

leaf

3.2

23456789

stem

Fig. 8. Polyphenol contents in an in vitro plant of Comus kousa Milky Way. (K. Ishimaru et aI. , unpubl.)

1 : gallic acid, 2: p-glucogallin, 3: 1, 2, 6-tri-O-galloyl-p-D-glucose, 4: 1, 3, 6-tri-O-galloyl-p-D-glucose,


5: 1,2,3, 6-tetra-O-galloyl-p-D-glucose, 6: 1,2,3,4, 6-penta-O-galloyl-p-D-glucose, 7: (+)-catechin,
8: (+)-gallocatechin, 9: procyanidin 8-3

ts'?

~ 16

to

.~ 2.4

:c

3.2,

6l
;:;

......

'"'

E::

o
o

:e

'"
B
o

~
1:;

'"

:::

1.6

1.4

0.8

1.0

R
9

0.0

0.0
4

0.2

0.2
3

0.4

~ 06

1.0

0.4

~ 0.6

"0

t'

_ 1.4
..:
.~ 1.2

~ 0.8

1.0

stem

0.8

"0

t'

:t

.~ 1.2

_
..:

1.6

1.6
1.4

root

1 : gallic acid, 2: Ji-glucogallin, 3: 1,2, 6-tri-O-galloyl-Ji-D-glucose, 4: 1,3, 6-tri-O-galloyl-Ji-D-glucose,


5: 1,2,3, 6-tetra-O-galloyl-Ji-D-glucose, 6: 1,2,3,4, 6-penta-O-galloyl-Ji-D-glucose, 7: (+)-catechin,
8: (+)-gallocatechin, 9: procyanidin B-3

leaf

1.8

1.8

Fig. 9. Polyphenol contents in an in vitro plant of Comus capitata Mountain Moon. (K. Ishimaru et aI., unpubl.)

0.0

0.2

0.4

~ 0.6

"0

t'

:t

.~ 1.2

..:

1.8

...::r
~

0>

.,'"'

V>

?"

'--'

I::l

Comus kousa (Dogwood)

123
Table 1. Effects of growth regulators on callus formation on leaf
segments of Comus kousa cultured on MS solid medium for 8
weeks. (Ishimaru et al. 1993)
2,4-D

NAA

IAA

BA

Callus fresh wt"


(mg)

0
0.1
0
0.1
0
0.1
0
0.1
0
0.1

50.9
240.4
137.9
179.5

(mg/l)
0.1
0.1
1.0
1.0
0.5
0.5
2.0
2.0
3.0
3.0

126.7
0
213
25.1
229.4

, Fresh weight: average for five samples.

glucose, is one of the lowest molecular weight compounds of galloylglucose


(hydrolyzable tannin), 9 is one of the most common procyanidins (condensed
tannin), while 7 and 8 are the structural components of condensed tannins.
Therefore, it was noteworthy that C. kousa calli produced both types
(hydrolyzable and condensed) of tannins.

3.2.3 Effects of Auxins on Growth and Tannin Production of


C. kousa Callus
To determine the growth and phenolic production (2 and 7-9) of the calli
on the media with different auxins, three media, A, B (0.5mg/1 NAA and
0.1 mg/l BA), and C (3mg/1 IAA and 0.1 mg/l BA), were used. The growth
rate of the calli cultured on these media is shown in Fig. 10. On these three
media, the amount of calli gradually increased throughout the culture period
(1 to 8 weeks). Especially the calli cultured on medium A showed the best
growth (44.4mg dry wt. / tube at week 8), a level almost double that on
medium B (21.6mg dry wt. / tube at week 8). For callus growth of, the
combination of 2,4-D and BA seemed to be superior to the others (NAA-BA
and IAA-BA).
The amounts of 2 and 7-9 produced in the calli cultured on media A-C are
shown in Fig. 11. The calli cultured on medium A contained a large amount
of 7 (Fig. llA), which rapidly increased from the early stage of the culture
and later reached the maximum level (230.9I1g / tube) at week 6; it decreased
until the end of the culture (8 week). Condensed tannin 9 was also observed
in relatively high content, with a level almost half that of 7. The amount of
9, after showing its highest level (153.9I1g / tube) at week 6, began to decrease similarly to that of 7. The level of 8 was almost one-fifth to one-third
of that of 7. Compound 2 appeared in a small amount throughout the culture
time.

124

K. Ishimaru et al.

200
------

:2 175

--u-

=r..~ 150

Fig. 10. Growth of callus (media A-C)


and cell suspension (medium A) cultures
of Comus kousa in the dark at 25C.
(Ishimaru et al. 1993)

callus on medium A
callus on medium B
callus on medium C
cells in medium A

-{J--

1:l

125

.a
-- 100
75

'ijl
~

50

t:25

O+---~~---r--'---~~---r~

Weeks

300

----A--

--

---0-

--0---

~ 250

.t:J.

.a

200

~
<

8
9

---0-

.a 200

-a 150
=
=
E

<100

50

50

--0---

250

100

----A--

.t:J.

!~~---"

-a 150
=
=
E

--

300

2
7

Weeks
300

-----A--

---0-

Qi
.t:J.

~
~

250

--0---

200

8
9

---0-

.,

--0---

80

II

c:

~60

~
...

-a 150
=
=
E

<

--2

--

100

2
7

8
9

/~~

2
7

Weeks
2
7

8
9

540

100

.---.

50

<

20

0
0

Weeks

0
6

Weeks

Fig. llA-D. Polyphenol production in calli (A-C) and cell suspension (D) of Comus kousa in the
dark at 25C. A On medium A. B On medium B. C On medium C. D On medium A. (Ishimaru
et al. 1993)

Comus kousa (Dogwood)

125

The calli cultured on medium B also produced similar phenolics (2 and 79) (Fig. lIB). In this culture, the amount of 7 increased from the early stage (1
week) and later reached the maximum level (130.3 Jig / tube) at week 4; it
rapidly decreased until the end (week 8) of the culture. The level of 9 appeared
remained steady (ca. 45 Jig / tube) during the first 4 weeks and after week 4 it
began to decrease. Compound 8, after showing its highest level (19.1 Jig / tube)
at week 2, also decreased continuously. At the end of the culture time (at week
8), these phenolics were not detected in the calli. Therefore, the combination
of NAA-BA was unsuitable for the production of phenolics in C. kousa callus
cultures.
In the calli cultured on medium C, the amounts of 2 and 7-9 gradually
increased from the early period of the culture to show their highest amounts
(2: 5.7 Jig, 7: 275.1 Jig, 8: 55 Jig and 9: 130.9 Jig / tube) at week 7 (Fig. 11 C). At
the last stage (7-8 weeks) of the culture, the simultaneous decrease of these
compounds was observed.
3.2.4 Suspension Cultures

C. kousa calli were also transferred into liquid medium A to establish cell
suspension culture. The growth rate of the cells is shown in Fig. 10. From the
early period of the culture the amount of the cells gradually increased and
after week 5 the increase was accelerated. The cells also produced phenolics
similar to those observed in the callus cultures, but their contents were fairly
low (Fig. lID). Compounds 7 and 9, which could be detected early in the
culture, suddenly decreased before week 3. Halfway (3-6 weeks) through the
culture, polyphenols did not appear at any detectable level. After week 6
the amounts of 7 and 9 increased slightly (9:28.4 Jig / flask at week 7, 7:15.3 Jig
/ flask at week 8), but, taking into account the good growth during this period,
the contents of these compounds in the cells were concluded to be very low.
Therefore, cell suspension culture of C. kousa in medium A was unsuitable for
the production of phenolics.
3.2.5 Effects of 2,4-D and BA Contents and Illumination on Growth and
Tannin Production of C. kousa Callus

To determine the effects of several combinations of 2,4-D-BA and illumination on the growth and tannin production of C. kousa calli, nine media, A
and D-K (Fig. 12), were prepared. On these media the illumination showed
no significant effect on callus growth except on media A, F, and K (Fig.
12A).
The production of phenolics (2 and 7-9) in calli cultured on these media
(A and D-K) under light or dark condition is shown in Fig. 13. In the light (Fig.
13A) the calli produced a sufficient level (over 30 Jig / tube of total amount of
2 and 7-9) of phenolics on four media F and I-K. The major compound
observed on these media was 9. On the other four media, D, E, G, and H, the

126

K. Ishimaru et al.

70
~

60

,Q

..

dark

30

.~ 30

20

t:

..

....

light
dark

r-

,Q

:: 40

60

--g!!

!>Il

..

70

0 light

50

50

!>Il

:c

40

~
~

10

20
10

0
A

medium
A
D
E
F

H
I

G
H
Medium

2,4-D

BA

0.1
0.5
1.0
0.1
0.5
1.0
0.1
0.5
1.0

0.1
0.1
0.1
0.5
0.5
0.5
1.0
1.0
1.0

G
H
Medium

(mg /I)

Fig. 12A-c' Growth of Comus kousa calli cultured on MS solid media with 2,4-D and BA for 5
weeks at 25C. A Basal MS. B Minus NH4N0 3 MS. (Ishimaru et al. 1993)

80

80

122 9

[Zl 9

70

G 8
.. 2

50

~60
~
!! 50

~40

~40

<:

<:

70

~60
,Q

!!

~ 8

o
..

7
2

=r o

~ 30

20

10

20
10

Medium

Medium

Fig. 13A,B. Polyphenol production in Comus kousa calli cultured on MS solid media with 2,4D
and BA for 5 weeks at 25C. A In the light B In the dark. (Ishimaru et al. 1993)

127

Comus kousa (Dogwood)

amount of 2 and 7-9 was fairly low. In the light, the addition of a high content
(1 mg/l) of BA (media I-K) seemed to be good for phenolic production. On
the other hand, in the dark (Fig. 13B), the calli produced high amounts of
phenolics on media A and F. The level of the total amount of 2 and 7-9 (ca.
34 j1g / tube) appearing on medium F was almost half that obtained on the
same medium under light. It was noteworthy that the amount of 9, observed in
high level on media I-K in the light, was very small on the same media in the
dark. This result suggested that illumination promoted the biosynthetic polymerization of 7 into 9 on these media (I-K). Except on the media with a low
content (O.lmg/l) of BA (media A and D-E), the light condition seemed to
have the greatest effect on polyphenol production in C. kousa calli.
3.2.6 Effects of NH4 N01 on Growth and Tannin Production of
C. kousa Callus

Nitrogen sources in culture medium are important factors for the production
of polyphenols in plant tissue cultures (Ishimaru and Shimomura 1991;
Ishimaru et al. 1992; Neera et al. 1992). In this experiment the effects of
NH 4N0 3 on the growth and polyphenol production of C. kousa calli were
determined. The growth of the calli cultured on nine media, A and D-K,
without NH 4 N0 3 is shown in Fig. 12B. In both light and dark conditions, callus
growth was much greater than that observed on the same media containing
NH4N0 3 (Fig. 12A). Especially, the amounts of the calli in the light were
almost 2.5 (on medium F) to 13 (on medium G) times larger than those on the
same media containing NH4 N0 3 . It was interesting that callus growth observed on the media without NH4NO} in the light was enhanced in proportion
to the concentration of 2,4-D (Fig. 12B).
The polyphenol production in the calli cultured on media A and D-K
(minus NH 4N0 3 ) was also observed to have considerably high level (Fig. 14).
1600
1400
~1 2oo

o
o

1600

1400

II 2

~1 2oo

'"

'"

fZl 9
D 8
0 7

II 2

.e woo

.&>

.e woo

.&>

~ 800

"5 600
e=
400

-==
o
e

200

200

0
A

F
G
Medium

Medium

Fig. 14A,B. Polyphenol production in Comus kousa calli cultured on MS solid media (minus
NH4 N0 3 ) with 2.4-D and BA for 5 weeks at 25C. A In the light. B In the dark. (Ishimaru et al.
1993)

128

K. Ishimaru et al.

The maximum amount of phenolics (ca. 1500,ug / tube of total amount)


observed on medium G without NH4N0 3 under light (Fig. 14A) was almost
over 20 times greater than that on medium F containing NH4N0 3 in the light
(Fig. 13A). On media A and D-H without NH 4N0 3, the illumination seemed
to significantly enhance the production of 9.
In the leaves of C. kousa plant (collected in June, in Japan), compounds 2
and 7-9 were also detected by HPLC analysis (2:0.017%,7:0.627%,8:0.222%
and 9:0.114 %, as dry wt.). The highest content of 7 observed in the intact plant
was comparable with that (0.609%, as dry wt.) of the same compound in the
calli cultured on medium A for 6 weeks (Fig. 11A).

4 Summary and Conclusion


Comus species, such as C. kousa chinensis, C. kousa Milky Way, C. kousa
Gold Star, C. kousa Satomi, C. kousa Snowboy, C. capitata Mountain Moon,
c. drummodii Eddie's White Wonder, and C. officinalis generally contain
hydrolyzable tannins in which the major component is 1,2,3,4,6-pentaO-galloyl-j3-D-glucose. In some varieties (+ )-catechin and related proanthocyanidin (condensed tannin) are also produced.
In three species, C. kousa chinensis, C. kousa Milky Way, and C. capitata
Mountain Moon, micropropagation through shoot proliferation, rooting in in
vitro, and transplantation to pots was successful. The tannin profiles in these
shoot cultures were different from those of the whole plant tissues; the major
compound observed in vitro plants was lowest molecular weight galloyglucose
j3-g1ucogallin. This result also suggested the suitability of shoot cultures of
Comus plants for the study of gallic acid metabolism.
Callus and cell suspension cultures of C. kousa produce phenolics
similar to those observed in the intact plant. Especially the contents of
flavan 3-01s and pro cyanidin were relatively high in the callus cultures. With
the determination of several culture conditions, C. kousa calli were clarified to
be usable for biosynthetic study of condensed tannins as well as for their
production.

5 Protocol
5.1 Shoot Cultures
Young branches were collected in summer and sterilized [70% ethanol (1 min), 1 % NaOel
(15 min), sterile water (rinse 2X)]. The axillary buds and shoot apices were cut off and aseptically
placed on BW medium under illumination (16-h photoperiod I a day, 3000 Ix). At 2-3-month
intervals, the shoots were subcultured on the same medium. For rooting, shoots (3-5 cm) were
transferred (after dipping of the cut ends into 70% EtOH with 20mM IBA for 1 s) on BW medium
containing 0.2mg/l NAA, 0.6mgll IBA, and 0.2% activated charcoal.

Comus kousa (Dogwood)

129

5.2 Callus and Cell Suspension Cultures


Leaf segments, collected in spring, were sterilized [70% ethanol (30 s), 2% NaOCI (1 Omin), sterile
water (rinse 3x) and aseptically placed on MS solid media (solidified with 2.S gil Gelrite)
supplemented with various combinations of 2,4-D, NAA, IAA, and BA. After 2 months of
culture, the calli derived on the three media A-C were separately transferred to the same medium
(A, B, or C) and subcultured at S-week intervals for over 1 year in the dark. The calli cultured on
medium A were also transferred into liquid medium A (SOml / 100mi flask) and the cell suspension culture was established. The cells were subcultured at 2-month intervals on a rotary shaker
(100 rpm) in the dark for 1 year.

5.3 Isolation of Phenolics


Fresh calli (61.9 g), cultured on medium A for S weeds in the dark, were macerated and extracted
with 90% aqueous acetone (200ml X 3) at room temperature. The extract was concentrated (to
ca. 20 ml) and applied to Sephadex LH -20 and Bondapak C18 Porasil B column chromatographies
(stepwise elution with H 20 and methanol) to give 2 (3.7 mg), 7 (12.1 mg), 8 (9.1 mg), and 9
(3.4mg).

5.4 Growth and Polyphenol Production of the Callus and Cell


Suspension Cultures
1. Callus cultures: piece of callus (ca. 0.1-0.3 g) was inoculated and cultured in a test tube [lOml
medium in one tube (2.2cm in the diameter) in the light (16-h photoperiod / a day, 3000 Ix) or
dark.
2. Cell suspension culture: ca. 0.7 g of cells were transferred and cultured in a flask (SOml
medium in 100mi flask, 100rpm) in the dark.

5.5 Quantitative Determination by HPLC


Lyophilized samples (20-30 mg) were crushed and extracted with 80% aqueous acetone or methanol (2 ml) for IS h at room temperature. Each extract, after filtration with a Millipore filter
(O.4Sj1m), was injected (S-ISj1I) into HPLC.
All cultures were placed at 2S dc.
Acknowledgments. This work was supported in part by the Physiologically Active Substances of
Trees Research Association (PASTRA), subsidy governed by the Forestry Agency, and by the
Ministry of Health and Welfare, Science Research Fund subsidy granted to the Japanese Health
Science Foundation.

References
Brinkman KA (1974) Comus L. Dogwood. In: Seeds of woody plants in the United States. USDA
Agric Handbood No 4S0, USDA, Forest Service, pp 336-342
Coker AL (1982) In vitro culture of flowering dogwood, Comus florida L. MS Thesis, Univ of
Tennessee, p 28

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Everett TH (1981) CORNUS, The New York Botanical Garden illustrated encyclopedia of
horticulture, vol 3, Garland, New York pp 873-878
Eyde RH (1988) Comprehending Comus: puzzles and progress in the systematics of the
dogwoods, Bot Rev 54:233-251
Haddock EA, Gupta RK, AI-ShaH SMK, Haslam E, Magnolato D (1982a) The metabolism of
gallic acid and hexahydroxydiphenic acid in plants. Part 1. Introduction. Naturally occurring
galloy esters. J Chern Soc Perkin Trans I 1982:2515-2524
Haddock EA, Gupta RK, Haslam E (1982b) The metabolism of gallic acid and
hexahydroxydiphenic acid in plants. Part 3. Esters of (R)- and (S)-hexahydroxydiphenic acid
and dehydrohexahydroxydiphcnic acid with D-glucopyranose CC 4 and related conformations). J Chern Soc Perkin Trans I 1982:2535-2545
Hatano T, Ogawa N, Kira R, Yasuhara T, Okuda T (1989a) Tannins of cornaceous plants I.
Cornusiins A, Band C, dimeric, monomeric and trimeric hydrolyzable tannins from Comus
officinalis, and orientation of valoneoyl group in related tannins. Chern Ph arm Bull 37:20832090
Hatano T, Yasuhara T, Okuda T (1989b) Tannins of cornaceous plants II. Cornusiins D, E and F,
new dimeric and trimeric hydrolyzable tannins from Comus officinalis. Chern Pharm Bull
37:2665-2669
Ishimaru K, Shimomura K (1991) Tannin production in hairy root culture of Geranium thunbergii.
Phytochemistry 30:825-828
Ishimaru K, Arakawa H, Neera S (1992) Tannin production in Liquidambar styraciflua callus
cultures. Plant Tissue Cult Lett 9:196-201
Ishimaru K, Arakawa H, Neera S (1993) Polyphenol production in cell cultures of Comus kousa.
Phytochemistry 32:1193-1197
Kashiwada Y, Nonaka G, Nishioka I (1984) Tannins and related compounds 23. Rhubarb (4):
isolation and structures of new classes of gallotannins. Chern Pharm Bull 32:3461-3470
Lee SH, Tanaka T, Nonaka G, Nishioka I (1989) Sedoheptulose digallate from Comus officinalis.
Phytochemistry 28:3469-3472
Linsmaier EM, Skoog F (1965) Organic growth factor requirements of tobacco tissue cultures.
Physiol Plant 18:100-127
Mitsuhashi H (1988) (ed) Comus officinalis Sieb. et Zucc., Illustrated medicinal plants of the
world in colour. Hokuryukan, Tokyo, 372pp
Murashige T, Skoog F (1962) A revised medium for rapid growth and bio-assays with tobacco
tissue cultures. Physiol Plant 15:473-497
Neera S, Arakawa H, Ishimaru K (1992) Tannin production in Sapium sebiferum callus cultures.
Phytochemistry 31:4143-4149
Niki T, Yoshida S, Sakai A (1978) Studies on chilling injury in plant cells I. Ultrastructural changes
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Niki T, Yoshida S, Sakai A (1979) Studies on chilling injury in plant cells II. Ultrastructural
changes in cells rewarmed at 26C after chilling trcatment. Plant Cell Physiol 20:899-908
Okuda T, Hatano T, Yasui T (1981) Revised structure of terchebin, isolated from Comus
officinalis. Heterocycles 16:1321-1324
Okuda T, Hatano T, Ogawa N, Kira R, Matsuda M (1984) Cornusiin A, a dimeric ellagitannin
forming four tautomers, and accompanying tannins in Comus officinalis. Chern Pharm Bull
32:4662-4665
Sato T (1991) Basic studies of organ and callus culture in woody plants. Bull For For Prod Res Inst
360:35-119
Thompson RS, Jacques D, Haslam E, Tanner RJN (1972) Plant proanthocyanidins. Part 1 Introduction; the isolation, structure, and distribution in nature of plant procyanidins. J Chern Soc
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Trigiano RN, Beaty RM, Dietrich JT (1989) Somatic embryogenesis and plantlet regeneration in
Comus florida. Plant Cell Rep 8:270-273
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YPS (ed) Biotechnology in agriculture and forestry, vol 20. High-Tech and micropropagation
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Yazaki K Okuda T (1993) Comus officinalis: In vitro culture and the production of gallotannins.
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Yoshida S, Tagawa F (1979) Alteration of the respiratory function in chill-sensitive callus due to
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IX Cynara cardunculus sUbsp.flavescens (Cardoon):


In Vitro Culture, and the Production of Cyprosins Milk-Clotting Enzymes
M.e. CORDEIRd, M.S. PAIS 2 , and P.E. BRODELIUS 1

1 Introduction
Flowers of Cynara cardunculus flavescens (cardoon) have been traditionally
used in Portugal and Spain for many centuries to produce artisanal cheeses.
One of the first reports on this use by farmers was made by Brotero (1804) in
Flora Lusitanica. Today, the Serpa and Serra cheeses are some examples of
typical and highly appreciated products of Portugal.
1.1 Taxonomy and Geographic Distribution

Taxonomically cardoon is included in the genus Cynara belonging to the


Asteraceae-Carduae family (Brotero 1804; Pereira Coutinho 1939; Tutin and
Heywood 1976; Franco 1984; Wiklund 1992). In a recent revision of the genus
Cynara, Wiklund (1992) includes the cardoon and the artichoke in the same
subspecies-C. cardunculus flavescens, as two different cultivars (Fig. 1). One
morphological difference between the two cultivars is that the cardoon is
prickly and the artichoke is not. Another subspecies also included in C.
cardunculus is subsp. cardunculus. These two subspecies appear to be geographically separated; subsp. flavescens occurs mainly in the Iberian Peninsula
and Macaronesian region while subsp. cardunculus has been found in the
central and northeastern Mediterranean region (Wiklund 1992).
In Portugal, cardoon (Fig. 2) grows spontaneously in the provinces of
Estremadura, Alentejo, and Algarve, and on Madeira. It grows in stony or
waste places and dry grassland, mainly on clay soils (Tutin and Heywood 1976;
Franco 1984).
1.2 Cheese-Making with Flowers of Cardoon

The common name of cardoon in Portuguese is cardo do coalho, which means


curdling cardoon. This name was given due to the usage of flowers by farmers
Department of Plant Biochemistry, Lund University, P.O. Box 117, S-22100 Lund, Sweden
Centro de Biotecnologia Vegetal, Faculdade de Ciencias de Lisboa, Bloco C2, Campo Grande,
P-1700 Lisboa, Portugal
1

Biotechnology in Agriculture and Forestry, Vol. 41


Medicinal and Aromatic Plants X (ed. by Y.P.S. Bajaj)
Springer-Verlag Berlin Heidelberg 1998

Cynara cardunculus subsp. fiavescens (Cardoon)

133

. . . - - - - - - - - - out-group (Cirsium vulgare)


. . . - - - - Cynara humilis
1 - - - - - Cynara cyrenaica

Cynara algerbiensis
Cynara comigera
. . . - - - - - - Cynara baetica
, - - - - - Cynara syriaca
. . . - - - Cynara auranitica
Cynara cardunculus subsp. cardunculus
Cynara cardunculus subsp. f1avescens - - [

cultivar Cardoon
cultivar Artichoke

Fig. 1. Tree of the genus Cynara with Cirsium vulgare as outgroup (After Wiklund 1992)

Fig. 2. Capitulum of Cynara cardunculus subsp. fiavescens cv. cardoon

to produce cheese. Many other species in over 13 genera are also commonly
referred to as cardo (= thistle) in Portugal (Pereira Coutinho 1939; Franco
1984).
Various cheeses made with flower extracts are still being produced in
Portugal and Spain by farmers or cheese-makers. There are two main types of
cheese in Portugal; the Serra cheese, made in the region of Serra da Estrela in
the center of the country, and the Serpa cheese made in Serpa, a village

134

M.e. Cordeiro et al.

situated in the District of Beja, in the south. The cheeses from Azeitao and
Castelo Branco also share characteristics with these cheeses. They are all
made with the flowers of cardoon, and with ewe's milk, only. The skills in
making these cheeses have been inherited over generations within the same
families. In Portugal, many families make their own cheeses for home consumption. Also in Spain various cheeses are made with extracts from Cynara
flowers. C. humilis is used to make many types of cheeses some examples are
Serena, Torta del Casar, Pedroches, and Grazalema.
Even though the cheeses are generally made in the same way, they show
distinct characteristics and differences in taste, depending on the region where
they are made. This is due to a number of factors that influence the ripening
process. The Serra cheese is a soft, creamy cheese and exhibits a very slight
and characteristic bitter or peppery flavor. The Serpa cheese shows a higher
curd firmness and has a stronger flavor.
Traditionally, the cheeses are made only with ewe's milk. The proteinases
are extracted from the flowers in two main ways: dried styles from flowers are
ground with salt in a mortar and added to the milk; or the styles are extracted
with lukewarm water (around 30C).
Farmers collect the mature flowers or the upper part of styles during June
and July and store them in dry places to be used for clotting of milk during the
following autumn and winter, when ewe's milk can be obtained.
When the cardoon flowers are collected in the fields, other flowers of
similar color and shape are also collected (e.g., Centaurea calcitrapa, Silybum
marianum, and other species of the genus Cynara). Although some of these
flowers also have some clotting activity, the proteolytic enzymes that are
present do not have the same properties as the cardoon proteases. Therefore,
they give rise to a different proteolysis of milk proteins, which results in
different characteristics and taste in the cheeses. Generally, the quality of
these cheeses will be reduced. Also, the stage of development at which the
flowers are collected will significantly influence cheese characteristics.
Another factor that may influence the standardization of cheeses is the
enzyme variation between populations of cardoon. At least three forms of the
Table 1. Review of in vitro culture studies on Cynara cardunculus

Study conducted

Remarks/observations

Reference

Cell cultures

Callus and suspension cultures


showing good growth were
established
Germination of embryos was
occasional; plantlets obtained
showed arrest of growth
No aspartic proteinase detected
Conditions for batch, semicontinuous,
and continuous culture systems
were established
No transient expression after
electroporation Transient gene
expression after using PEG

Figueiredo et al. (1987)

Somatic embryogenesis

Proteases in cell cultures


Cells in bioreactors

Transient gene expression


in protoplasts

Miguel and Pais (1993)

Cordeiro et al. (1993)


Costa( 1994)

Carocha et al. (1994)

Cynara cardunculus subsp. fiavescens (Cardoon)

135

enzyme exhibiting different clotting characteristics appear to be present in


cardoon flowers (Heimgartner et al. 1990). Flowers collected in different
regions in the provinces of Alentejo and Algarve in Portugal have shown
different clotting and proteolytic activities due to different relative amounts of
these various forms of the enzyme (Cordeiro et al. 1997). Consequently, for
large-scale production, a standardization of the enzyme used is very important
to maintain the quality and characteristics of cheeses made with the cardoon
protease. A review of the present situation of producing such cheeses has been
published (Bivar Roseiro 1991).
1.3 Secondary Metabolites

Various secondary metabolites have been isolated from Cynara species. In


particular, phenolics and terpenoids have been found. C. scolymus is a source
for the choleretic compound cynarin (1,5-dicaffeoyl-quinic acid). Germinating
seeds of C. scolymus contain considerably more cynarin than leaves (Ben-Hod
et al. 1992). A variety of flavanoids and flavanoid glycosides have been
extracted from C. scolymus (EI-Negoumy et al. 1987; Hinou et al. 1989;
Hammouda et al. 1993) and C. sibthorpiana (EI-Ansari et al. 1988). The globe
artichoke also contains polyacetylenes (Stevens et al. 1990). From C.
cardunculus a number of sesquiterpene glycosides and saponins (Shimizu et al.
1988), and from C. humilis some sesquiterpene lactones and guaianolides have
been isolated (Reis et al. 1992).

2 In Vitro Culture Studies


2.1 Cell Suspension Cultures of C. cardunculus

Callus and suspension cultures of C. cardunculus were first established


by Figueiredo et al. (1987). The explant was taken from hypocotyls of
germinated seeds. Growth characterization was performed in B5 medium
supplemented with plant growth regulators (1 mg/l 2,4-D and 0.1 mg/l
BA), and in TNO, - medium supplemented with 1 mg/l 2,4-D and 0.1 mg/l
kinetin. Different growth patterns were obtained in these two media. The
B5 medium is more adequate for biomass production, while the other
medium promotes synthesis of proteases (Cordeiro et al. 1993). These
proteases were characterized and compared to the flower enzymes. When
compared to the flower cyprosins, the specific proteolytic and milk-clotting
activities of the cell proteases is much lower. Analysis by Western or Northern
blots has shown that the flower-specific cyprosins are not expressed in these
suspension-cultured cells (Cordeiro et al. 1993). Furthermore, characterization
with specific protease inhibitors shows that the culture proteases are mostly
inhibited by cystatin and leupeptin, indicating that they are cysteine proteases.

136

M.e. Cordeiro et al.

Two other lines of cell cultures were obtained from tissues of young flowers
(unpubl.). Both these cultures were grown in B5 medium using hormone
concentrations similar to those given above. Western blot analysis and clotting
experiments have shown that these cell lines also do not synthesize cyprosins
under the conditions used (V. Carocha, M.e. Cordeiro, and M.S. Pais, unpubl.).
Cyprosins are present mostly at later stages of flower development, and
accumulate in the styles of these flowers, indicating that the synthesis of the
enzymes is related to differentiated tissues and cells. This is not the case with
cells in suspension cultures, which generally are undifferentiated.
All these cell suspension cultures may thus be used as recipient cells for
the genes encoding the cyprosins. Such transgenic cell cultures will be of great
importance in studies on posttranslational modifications of cyprosins and for
the production of cyprosins on a large scale.
2.2 Transient Gene Expression in Cardoon Protoplasts

Cell suspension cultures derived from young flower tissues were used to isolate
protoplasts. Conditions for cultured protoplasts to regenerate cell walls and
callus cultures have been established (unpubl.). Initial studies have been performed to obtain appropriate conditions for gene transfer into protoplasts.
Direct gene transfer was attempted using electroporation and polyethylene
glycol 4000 (PEG), and using the pDW2 plasmid (Pietrzak et al. 1986). This
plasmid contains the CAT (chloramphenicol acetyl transferase) reporter gene
and the 35S cauliflower mosaic virus promoter. Under the conditions studied,
no CAT activity was detected when electroporation was tested. Transient gene
expression was obtained when using 30% PEG and without the heat-shock
treatment prior to the addition of DNA and PEG (Carocha et al. 1994). These
studies suggest that direct gene transfer is possible in C. cardunculus and will
be continued to establish stable transformation using cyprosin genes.
2.3 Somatic Embryogenesis in C. cardunculus

Somatic embryos were produced from cotyledons and first leaves of in vitrogrown seedlings of C. cardunculus (Miguel and Pais 1993). Embryogenic calli
were obtained after 4-5 weeks of culture on B5 solid medium. Cotyledon
explants presented a higher percentage of embryogenic induction than leaf
explants. In the presence of 1 mg/l 2,4-D and 0.1 mg/l kinetin, initial stages of
somatic embryo development were formed on the surface of embryogenic
calli. Transfer of these cultures to B5 liquid medium and a different hormone
concentration (zeatin 1 mg/l and 2,4-D 0.1 mg/l) allowed the induction of more
embryos and their further development (Fig. 3). Mature embryos, when transferred to the same liquid medium without growth regulators, showed root
apex growth and greening of cotyledons (Fig. 3). However, the germination of
the embryos was still only occasional and plantlets obtained showed arrest of
growth before development of leaves (Miguel and Pais 1993).

Cynara cardunculus subsp.fiavescens (Cardoon)

137

Fig. 3A-E. Embryonic cell cultures of Cynara cardunculus. A Group of somatic embryos at

various developmental stages, including early cotyledonary stages, formed on embryogenic callus.
8-E Cultures in liquid medium. 8 Embryoids that remained attached to the cotyledon explant. C
Torpedo stage. D Initiation of root apex growth . E Mature embryos with large cotyledons. Bars
1 mm. (Miguel and Pais 1993)

2.4 Cell Suspension Cultures in Bioreactors


C. cardunculus cell suspension cultures were used as a model system to study
the effects of hydrodynamic stress on cell viability, growth kinetics, and
protease synthesis in a 2-1 mechanically stirred bioreactor (Costa 1994). Low
oxygen mass transfer coefficient (kLa > 14h- 1) has a negative effect on cell
growth and metabolic activity. However, comparison of characteristic times of
metabolic processes showed that oxygen is not a limiting nutrient, even at low
agitation conditions (30rpm).
The growth rate of cell suspension cultures was studied in discontinuous,
semicontinuous, or continuous culture systems. The influence of essential
macronutrients (carbon, nitrogen, and phosphorus) was studied in a discontinuous system. Low concentrations of Pi in the growth medium restrict cell
growth and reduce the protease activity of cultured cells.
Continuous cell culture of C. cardunculus could be successfully established by using a chemos tat. The culture presented good growth characteristics
due to a high ability to adapt to new environmental conditions. Low aggrega-

138

M.e. Cordeiro et al.

tion and low cell growth on the walls of the fermenter were advantageous
properties of C. cardunculus cell suspensions. Proteases (not characterized)
were produced in B5 medium. In continuous culture, a higher protease level
corresponded to a higher specific growth rate of the cells, which showed that
the synthesis of proteases is linked to primary metabolism (Costa 1994).

3 Isolation of the Milk-Clotting Enzymes


The purification and partial characterization of the proteases from the flowers
of C. cardunculus was first reported by Heimgartner et al. (1990). Three
enzymes with milk-clotting activity were isolated from dried flowers. The same
enzymes may be extracted from fresh flowers. Extraction of proteins from
flowers was carried out at pH 8.3 at which the enzymes are essentially inactive,
preventing any autohydrolysis during purification. The purification procedure
included fractionation with ammonium sulfate precipitation (30-80% saturation), anion exchange chromatography on DEAE-Sepharose, and Mono QFPLC.
The name cynarase was first given to the three purified enzymes, cynarase
1, 2, and 3. It was derived from cynara protease. Later, according to the
catalytic properties of the enzymes, and due to conventional rules on naming
aspartic proteinases, this name was changed to cyprosin. This was also derived
from cynara protease, adding the sin ending.
Two aspartic proteinase, cardosin A and B, were extracted from fresh
flowers of a different variety of cardoon at acid pH (3) (Faro et al. 1992). At
this acidic pH the enzymes are active and autohydrolysis during purification is
possible.

4 Characterization of the Enzymes


Molecular Weights. The enzymes are heterodimeric proteins and the subunit
sizes, as estimated by SDS-PAGE, are 32.5 + 16.5kDa, 33.5 + 16.5kDa, and
35.5 + 13.5kDa for cyprosin 1,2, and 3, respectively (Fig. 4). A native Mr of
around 49 kDa was determined for the three cyprosins by native PAGE. Gel
filtration chromatography indicated somewhat lower native Mrs, i.e., 41, 42,
and 45 kDa for cyprosin 1, 2, and 3, respectively. The cardosins are also
heterodimeric enzymes and the subunit sizes are 31 + 15 kDa (cardosin A) and
34 + 14kDa (cardosin B) (Faro et al. 1993).
Immunological Properties. Polyclonal antibodies (rabbit), raised against
the large subunit of cyprosin 3, react with the large subunits of the other
two cyprosins, indicating some common features of the three forms of the
enzyme.

Cynara cardunculus subsp. fiavescens (Cardoon)

139

Fig. 4A,B. SDS-PAGE of crude extracts


(panel A) and purified cyprosins (panel B)
from flowers of C. cardunculus. Lanes 1-4
Crude extracts from violet, upper white,
lower white part of fresh flowers, and dried
flowers, respectively; lanes 5-7 purified
cyprosin 1,2, and 3, respectively; lane 8 M,
standards. (Heimgartner et al. 1990)

kDa
94

67

43

30

'.

20

14.4

Isoelectric Points. Electrophoretically pure cyprosin 2 and 3 were run on twodimensional electrophoresis gels (IEF and SDS-PAGE) and were both separated into three isozymes with very close isoelectric points, i.e., 3.S5, 4.00, and
4.15 (Cordeiro et al. 1994b).
Preparative isoelectric focusing under nondenaturing conditions was used
to purify the three isozymes of cyprosin 3 (the most active isozyme) for further
studies of this isozyme (Cordeiro 1993).
Peptide Mapping and Protein Microsequencing. From the results summarized
above, it may be concluded that the natural population of cyprosins in flowers
of C. cardunculus is very complex. They vary in subunit size and they exist in
different isoforms. Electrophoretically (SDS-PAGE) purified subunits of
cyprosin 2 and 3 could not be directly sequenced due to this heterogeneity
(Cordeiro et al. 1994b). In an attempt to overcome this difficulty, peptides of
the subunits of these isozymes were produced by enzymatic (trypsin) or chemical (BrCN) cleavage, and electrophoretically purified.
The results obtained indicate that the various forms of cyprosins present
in flowers of cardoon have common structural features and that they may be
derived from common procyprosins. The occurrence of heterodimeric forms
of the enzyme containing subunits of different sizes may be due to proteolytic
cleavage of a procyprosin at different sites originating a group of closely
related enzymes. The occurrence of isozymes may be due to the expression of
a gene family, as discussed below (Sects. 5.1 and 5.4).
An internal partial N-terminal sequence was obtained from a BrCNpeptide obtained by cleavage of the large subunit of cyprosin 2 (Cordeiro et al.
1994b). This internal sequence (met-Ieu-asn-gln-gly-Ieu-val-gln-glu) has been
of great importance for the identification of a cDNA clone coding for cyprosin
(Cordeiro et al. 1994a).
Also an N-terminal sequence of purified cyprosin 1 has been obtained.
This sequence indicates that the N-terminus of the mature protein is asp-ser-

140

M.e. Cordeiro et al.

asp-leu -ala -val-val-ala -leu -thr -asn -asp-X -asp-thr -X -tyr-phe-gly -X -ile-gly
(unpubl.).
Internal sequences of cardosin A (ser-glu-glu-Ieu-gln-val-asp-asn-asn-thrleu-ser-X-met-pro) and B (ser-ala-glu-asp-ile-val-asn-asn-asn-gly-ile-ser-Xmet-pro) have been reported (Faro et al. 1993). These sequences cannot be
found in the deduced amino acid sequences of the two cyprosin cDNA clones
isolated in our laboratories (see below). This is a strong indication that the
cyprosins and cardosins are isolated from different Cynara species or cultivars.
Glycosylation. Endoglucosidase H treatment of cyprosins and subsequent
SDS-PAGE has shown that all three forms of the enzyme contain high
mannose-type glycans (Heimgartner et al. 1990). We also have indications that
a second glucan not removed by endoglucosidase H is bound to the cyprosins.
This assumption is also supported by the fact that the deduced amino acid
sequence of two cyprosin cDNAs each contain two putative glycosylation sites
(Cordeiro et al. 1994a; M. Pietrzak et al., unpubl.).
pH Optimum of Cyprosins. A synthetic peptide (lys-pro-Ieu-gln-Ieu-nph-argleu) was used to estimate the pH optimum of the three cyprosin 3 isozymes.
The pH optimum (4.1) was the same for the three isozymes (Brodelius et al.
1995).
Catalytic Properties and Substrate Specificity. Initially, the proteolytic activity
of various enzyme preparations was determined with a standard flu oro metric
proteolytic assay based on the release of TCA soluble FITC-Iabeled peptides
from casein (Heimgartner et al. 1990). Using this assay we could establish that
cyprosin 3 showed the highest specific activity and cyprosin 1 the lowest.
Furthermore, we could establish that the cyprosins belong to the aspartic
proteinase family. Addition of pepstatin A to the assay mixture inhibited the
activity of all three cyprosins almost completely, while other protease inhibitors such as leupeptin, aprotenin, cystatin, and iodoacetamide did not affect
the activity to any great extent.
A "library" of related chromophoric octa-peptide substrates with systematic variation in the amino acid residues has been used to study the proteolytic
activity of three cyprosins (pI 3.85, 4.00 and 4.15; Cordeiro et al. 1997). The
rate of hydrolysis of 46 peptides was determined to investigate the effects of
various substitutions in the PS-P j and the PZ'-P3 ' positions on enzyme activity.
From these experiments it is evident that cyprosin, like other aspartic
proteinases, preferentially cleaves peptide bonds between two hydrophobic
amino acids.
Four peptides were selected for detailed kinetic measurements. The
lowest Km values (15-25 mM) were obtained with lys-pro-ile-val-phe-nph-argleu while the highest Kcat values (34-85 S-I) were obtained with lys-pro-ile-Ieuphe-nph-arg-Ieu. The Kj-values for pepstatin A were below 0.1 nM for the
three isozymes.

Cynara cardunculus subsp.fiavescens (Cardoon)

141

Proteolytic and Clotting Activity. Hydrolysis of milk proteins by cyprosins was


investigated by determining the release of non-protein-nitrogen (NPN) from
milk. Clotting activity was followed with a formagraph using cow's or ewe's
milk. The pattern of breakdown products after hydrolysis of milk proteins
was determined by ultra thin isoelectric focusing (UTIEF). All these studies
were performed in comparison with chymosin, which is extracted from the
abomasum of calves and is the most-used enzyme in industrial cheese-making.
Chymosin has a very specific activity on milk proteins; it cleaves only one
bond, Phe IOS -Met I06 , in K casein.
In this comparison, purified cyprosin 3 and chymosin revealed similar
clotting activities (Fig. 5), in particular in the initial phase of the hydrolysis
when curd is formed (Fig. 6; Cordeiro et al. 1992). Slight differences in curd
firmness were detected which after the ripening period give rise to a softer
cheese made with the plant enzyme. NPN and UTIEF studies with cow milk
indicated that the cyprosins preferentially cleaved the phe lOs -met I06 -bond of K
casein. However, in addition to this protein, cyprosins also slowly hydrolyzed
some of the },-caseins, fJ-casein A 2, and asl-casein. Cyprosin 3 has shown a
considerably higher clotting activity in ewe's milk than chymosin, and also a
more specific hydrolysis of proteins in this type of milk (Cordeiro et al. 1992).

80

70

60
~

c:

E 50

:::E
i=

z 40
2
....

CD

:::J

30

20

10

Fig. 5. Formagram from clotting assays. AlO Curd firmness; r clotting time; r + 10 clotting time +
lOmin. Samples were: A and B cyprosin 3 (60,ug/assay); C and D chymosin (94,ug/assay); E
cyprosin 2 (53,ug/assay); F cyprosin 2 (106,ug/assay); G cyprosin 1 (160,ug/assay); H cyprosin 1
(320,ug/assay). (Cordeiro et al. 1992)

142

M.e. Cordeiro et al.

460
440
420

S
Q.,
Q.,

400
380

'-'

Z
~
Z

,,

360

chymosin

400

340

360
320
320
300

--

280

280
0

200

10

400

20

30

600

40

800

1000

1200

INCUBATION TIME (min)


Fig. 6. NPN concentrations as function of incubation time after addition of enzyme to milk from
cow. The concentration for proteins was for chymosin 9.4, for crude extract 73, and for cyprosin
3 12.ug/ml milk. (After Cordeiro et al. 1992)

5 Primary Structure of Cyprosin


5.1 Cyprosin eDNA Clones

A cDNA library was constructed from mRNA isolated from young flowers of
cardoon. Six clones were selected in the first screening using cyprosin antibodies and were named cyprol to cypro6. Cypro4 and cypro6, giving the
strongest signal in the screening, were sequenced but no homology to other
aspartic acid proteinases could be established. Clone cyprol actually contained two EcoRI fragments (2.0 and 1.7kb). The subclone containing the 1.7kb insert was called cyprols and sequenced. The deduced amino acid sequence
of cyprols contained the internal peptide sequence obtained by sequencing a
BrCN-fragment of cyprosin 2, showing that this cDNA clone encodes a
cyprosin.
In a second screening of the cDNA library using cyprols as probe, five
new clones named cypro7 to cyproll were selected. Cyproll (1.8kb) was
selected for sequencing.
The nucleotide sequence of clone cyprols contains a 1422-bp open reading frame coding for 474 amino acids including a putative full-length mature
protein (440 amino acids) and a partial prosequence (34 amino acids). By
PCR, a fragment corresponding to the missing 5' -end of this clone could be

Cynara cardunculus subsp. fiavescens (Cardoon)

143

isolated from the cDNA library and sequenced. The full-length gene product
contains 505 amino acids. The nucleotide sequence of clone cyproll contains
a 1527-bp open reading frame coding for 509 amino acids, including a putative
full-length mature protein (441 amino acids) and a prosequence (68 amino
acids).
5.2 Comparison of Deduced Amino Acid Sequence of Cyprosin to Other
Aspartic Proteinases

The deduced amino acid sequences of the two cyprosin cDNA clones are
shown in Fig. 7 in comparison with other plant aspartic proteinases. They are
called cyprosin I (cyprols) and II (cyprolJ), since it has not yet been firmly
established which form of the enzyme corresponds to which cDNA clone.
They show a relatively high homology to each other and to the other plant
aspartic proteinases, as indicated by the consensus sequence. Compared to
other aspartic proteinases, the plant enzymes contain an insert of around 100
amino acids (plant-specific insert; italics in Fig. 7) in the C-terminal part of the
protein molecule. Omitting this insert, the homology to aspartic proteinases
from other phyla is relatively high. For instance, the mature cyprosin I shows
34, 43, and 55% identity to rhizopuspepsin, bovine chymosin b, and human
cathepsin D, respectively.
Two putative active-site aspartic acid residues (DTG and DSG) have been
identified in each isozyme (bold and italics in Fig. 7). Furthermore, two putative glycosylation sites have been found in each cyprosin isozyme (underlined
in Fig. 7). One of these glycosylation sites, present in the plant-specific insert,
is conserved in all plant enzymes. It is well established that the cyprosins are
glycoproteins (Heimgartner et al. 1990). Furthermore, 12 conserved cysteins
are found in the plant aspartic proteinases (marked with * in Fig. 7).
A consensus sequence based on 30 aspartic proteinases (not shown) shows
that the mature aspartic proteinases are highly conserved proteins. In particular, the sequences around and at the active-site aspartyl residues are very
conserved. An exception to this is observed for the plant enzymes in which the
threonine of the second active site has been substituted for by serine. However, in spite of this substitution, the residues around the aspartyl residues of
the active site are conserved in the plant enzymes.
From a phylogenetic analysis (not shown), it may be concluded that the
plant enzymes are most closely related to the cathepsin D enzymes of various
animals.
5.3 The Plant-Specific Insert

A plant-specific region is well identified in the plant enzymes included in the


alignment in Fig. 7 (italics). The size of these inserts is 99-104 amino acids for
the various enzymes. The size of the mature aspartic proteinases of plant
origin is, therefore, somewhat larger (around 440 amino acids) than that of
aspartic proteinases from other phyla (325-360 amino acids).

hDeMCtmCqM
hDeMCtfCEM
aDpMCsaCEM
gDaaCsaCEM
sDaMCsvCEM
sgpMCnaCEM
-D-MC--CEM

GPLWILGDVF
GPLWILGDVF
GPLWILGDVF
GPLWILGDVF
GPLWILGDVF
GPLWILGDVF
GPLWILGDVF

kskGK.Ssgl
ennGKSSsgv
dEpvKSnglr
kEnaK1Sngv
kEn ..... 1g
dEaGeSng1q
-E-GKSS---

401
FTAMDvaPPh
FTAMDvaPPR
FTAMDipPPR
FiAlDvaPPR
FmAfDipPPR
FTAMDipPPR
FTAMD--PPR

GFCAsGCAAI
GFCsdGCAAI
GFCAgGCAAI
GyCesGCsAI
GFCAkGCAAI
GFCAsGCsAI
GFCA-GCAAI

201
GDVLIGdKtT
GDVLledKtT
GDVLvGGKsT
GDVLIGGapT
GDlLldGhsT
GDVLIGGKtT
GDVLIGGK-T

EPGITFlaAK
EPGITFlaAK
EPGITFlvAK
EPGITFvlAK
EtsvTFiigK
EPGlTFmvAK
EPGITF--AK

101
KeQdFIEATK
KeQdFIEATK
KdQeFIEATK
KdQeFIEATK
KnQkFIEATr
KdQeFIEATK
K-Q-FIEATK

301

NYMdAQYfGE
NYMdAQYyGE
NYMnAQYfGE
NYldAQYyGE
dYlntQYyGv
NYMnAQYfGE
NYM-AQY-GE

1
dsdgeliALK
dsgsDliALK
seeegDIVALK
sgdaDIVtLK
ssdsDpVpLv
ggegDIVALK
-----DIVALK

MGqYHTVFDY
MGrYHTVFDY
MGpYHTVFDY
MGkYHTVFDf
MGaYHTVFDf
MGaYHTVFDY
MG-YHTVFDY

AVVWMQNQir
AVVWMQNQik
AVVWMQNQLa
AvvwiQsQLr
AVVWieNQLr
AVVWMQNQLa
AVVWMQNQL-

ADSGISLLAG
ADSGISLLAG
ADSGISLLAG
ADSGISLLAG
vDSGISLLAG
ADSGISLLAG
ADSGISLLAG

FDGILGLGFQ
FDGILGLGFQ
FDGILGLGFk
FDGILGLGFQ
FDGILGLGyp
FDGILGLGFQ
FDGILGLGFQ

IGiGTPPQKF
IGiGsPPQKF
IGvGTPPQKF
IaiGTPPQKF
IG1GsPPQnF
IGvGTPPQKF
IG-GTPPQKF

YVdkLCeRLP
YVNeLCdRLP
YVNqLCnRLP
YVNeLCrRip
YaNqLCeRLP
YiNqLCdkLP
YVN-LC-RLP

AIGAaGVmSQ
AIGAkGVmSQ
kIGAaGVvSQ
AIGAsGVaSQ
AIGAeGiiSt
kIGAtGVvSQ
AIGA-GV-SQ

WYtMlnQGLV
WYnMvnQGLV
WYkMieQGLV
WYnMlkQGLy
WqsMqeQeLl
WYkMveQGLV
WY-M--QGLV

LWVPSsKCYF
LWVPSAKCYF
LWVPSAKCYF
LWVPSsKCYF
LWVPSAKCYF
LWVPSAKCYF
LWVPSAKCYF

441
GnlRVGFAeA A
GK1RVGFAeA A
GK1RiGFAkA A
GKaqVGFAeA A
GKdRiGFAks A
GKmRVGFAks A
GK-RVGFA-A A

rNeTedn1iN
QNkTqd11Ld
QNmTqer1Ld
eNkTke1ILN
QNkTqd11LN
QN-T---ILN

Q~een1iN

tTtlvTqINq
PTAIITeINh
PTAIITeINe
PTtIITmINh
PTAlvaqvNh
PTAIITeINe
PTAIIT-IN-

EISVGdAVPV
EISVGksVPl
EISVGkAVPV
EISVGnAaPV
EISVGkApPi
EISVGdAVPV
EISVG-AVPV

TVIFDTGSSN
TVIFDTGSSN
TVIFDTGSSN
TVvFDTGSSN
TVIFDTGSSN
TVIFDTGSSN
TVIFDTGSSN

SPMGESAVDC
SPMGESAVDC
SPMGESAVDC
SPMGESAVDC
SPnGEStVsC
SPMGESsVDC
SPMGESAVDC

qCKs1VdQYG
qCKT1VSQYG
eCKTiVSQYG
qCKTvVdQYG
eCKevVSeYG
eCKTvVSQYG
-CKT-VSQYG

qEPVFSFWLN
qEPVFSFWfN
sdPVFSFWLN
kEPVFSFWLN
addVFSFWLN
sEPVFSFWfN
-EPVFSFWLN

SvAC1FHSkY
SvAClFHSkY
SIACylHSrY
SIAC1FHSkY
SIACylHSrY
SIACfFHSrY
SIAC-FHS-Y

sSLSSMPnla
nSLSSMPnla
gSLgSMPdle
aqLStMPtvs
hqiSkMPnla
gSLaSMPeIs
-SLSSMP-I-

ksmiemLLsE
ktmiemLLsE
qq1LdLLLaE
qt1LdLLLsE
em1LnLLiaq
qq1LdLLLaE
--1L-LLL-E

RnaDEqEGGE
RnaDEeEGGE
RhvDEgEGGE
RnaDdeEGGE
RdpDassGGE
RhsDEgEGGE
R--DE-EGGE

rStdStTYKK
KSshSSTYKK
KagaSSTYKK
KSsrSSTYeK
nSkkSSsYKa
KSgqSSTYqK
KS--SSTYKK

FTvGGKtFnL
FTIGGKvFeL
FTIGGKkFaL
lTIGGKvFdL
FTlanKtFiL
FTIGaKkFaL
FTIGGK-F-L

eQPeK1CSQm
aQPdK1CSQm
TQPkK1CSQv
TQPkK1CSQi
TdPqKvCSQv
TQPsK1CSQv
TQP-K1CSQ-

LVFGGvDPnH
LVFGGvDPnH
iiFGGmDPkH
LVFGGvDPkH
LVFGGmDPkH
iVFGGmDPsH
LVFGG-DP-H

NGKsAAIQYG
NGtsAAIQYG
NGKpAAIQYG
NGKsAAlhYG
dGetckltYG
NGKpAAIQYG
NGK-AAIQYG

sPEqYvLKVG
cPEqYILKiG
kPEeYILKVG
aPheYvLKVG
tPEqYlvKle
kPEeYILKVG
-PE-YILKVG

kLCsFDGshd
kLCTFDGaRd
GLCTFDGtRg
GLCTFDGkRg
GLCmFDGkRs
GLCTFDGkhg
GLCTFDG-R-

fKGeHTYVPV
fKGkHTYVPV
yvGeHTYVPV
fKGqHiYVPV
yKGdHTYVPV
yKGnHTYVPV
-KG-HTYVPV

TGSIsGFFSq
TGSIsGFvSq
TGSlaGyFSe
TGalaGFFSn
sGaIsGFFSk
TGSlaGFFSe
TGSI-GFFS-

400
EGAtAQCISG
EGeAAQCISG
EGAAAQCISG
EGAAAQCISG
qGgqtvCISG
EGAAAQCISG
EGAAAQCISG

tSmi1ESVVD
aSsi1ESVVD
VSaG1rSVVD
VSmGIESVVD
VSnG1ESVVD
VSaG1kSVVD
VS-GIESVVD

300

200
TQKGYWQFeM
TeKGYWQFdM
TQKGYWQFdM
TQKGYWQFdM
srKGYWQFnM
sQKGYWQFeM
TQKGYWQF-M

100
DSVklGDL1V
DSVklGDLVV
DSVtVGDLVV
DaVtVGDLVV
DnV1VGDqVV
DSVtVGDLVV
DSV-VGDLVV

1: D12777; rice 2: D32144)

Fig. 7. Alignment of deduced amino acid sequences of six plant aspartic proteinases. The putative mature proteins are shown. The consensus sequence is
based on identical amino acid residues (upper case) in at least four of the six enzymes. Amino acid residues identical for all six plant aspartic proteinases
are shown in bold. Putative active site aspartic acids are in italic and bold. Putative glycosylation sites are underlined. The plant-specific insert is shown in
italic. Conserved cysteine residues are marked*. (EMBL Data Bank: cyprosin I: X69193; cyprosin II: X81984; barley AP: X56136; brassica AP: X77260; rice

Consensus

cyprosin I
cyprosin II
barley AP
bras sica AP
rice 1
rice 2

Consensus

cyprosin I
cyprosin II
barley AP
bras sica AP
rice 1
rice 2

Consensus

cyprosin I
cyprosin II
barley AP
brassica AP
rice 1
rice 2

cyprosin I
cyprosin II
barley AP
brassica AP
rice 1
rice 2
Consensus

Consensus

cyprosin I
cyprosin II
barley AP
brassica AP
rice 1
rice 2

?'-

(1)
::;.

0-

...,0

.j>.

.....
.j>.

Cynara cardunculus subsp.fiavescens (Cardoon)

145

From the consensus sequence in Fig. 7, it may be calculated that the plantspecific insert shows 73% conserved residues, indicating that this region has
similar characteristics. It is interesting to note that the six cysteine residues
present in the plant-specific region are all conserved in the six enzymes,
indicating a possible conserved tertiary structure of this region through
disulfide bridges. Furthermore, a putative glycosylation site (underlined in Fig.
7) is conserved in the plant-specific region in all six enzymes. Due to these
conserved features of the plant-specific insert, it may be assumed that this
additional part of the protein has a specific function in the plant. At present,
we can only speculate about this function.
5.4 Genomic Organization of Cyprosin Genes

Genomic DNA from young flower tissue of C. cardunculus was isolated,


digested with restriction endonucleases (EcoRI and HindIII) , and analyzed by
Southern hybridization (Cordeiro et al. 1994a). The EcoRI and HindIII digests each showed four to five strong hybridizing bands and several minor
bands when the entire 1.7-kb insert of cyprols was used as probe. From these
hybridization patterns it may be suggested that the cyprosin genes are organized as a multigene family.
The simultaneous expression of a number of cyprosin genes can explain
the heterogeneity observed in the gene product described above. This assumption is supported by the fact that two distinctly different but closely related
cyprosin cDNA clones have been isolated.

6 Modeling the Structure of Cyprosin


The crystal structure of a number of aspartic proteinases has been solved at
high resolution (Tang and Wong 1987; Davies 1990). The crystal structures of
these aspartic proteinases are closely related in overall shapes and the tracing
of chain foldings even though the amino acid homology is low in some cases.
Because of the similarities in primary and tertiary structures, it has been
possible to model the tertiary structures of aspartic proteinases by fitting
the amino-acid sequences onto existing, presumably homologous, crystal
structures.
A three-dimensional model of cyprosin has been proposed (Cordeiro et
al. 1994c) which was constructed using the deduced amino-acid sequence of
cyprols cDNA and which was based on the three-dimensional structures of
homologous aspartic proteinases.
The 3-D model of the enzyme was constructed using the rule-based comparative modeling approach encoded in the COMPOSER suite of computer
programs (Blundell et al. 1987; Sutcliffe et al. 1987a,b). Six highly homologous
aspartic proteinases were used for modeling, i.e., human cathepsin D, yeast
proteinase A, bovine chymosin, human renin, mouse renin, and porcine pep-

146

M.e. Cordeiro et al.

Fig. 8. Schematic representation showing the overall "fold" of cyprosin, generated using the
program 0 (Jones et al. 1991). Helices are shown as cylinders and sheets as flat arrows .
The remaining polypeptide chain is shown as a rope which represents the turn/coil regions in the
molecule. Numbers of amino acid residues are according to Fig. 6. The location of the plantspecific insert is indicated by a gap between G246 and S351. The catalytic aspartate side chains
(D35 and D222) and conserved cysteins participating in disulfide bridges (C48-C54, C213-C217,
C360-C397) are also shown and labeled. (Cordeiro et al. 1994c)

sin. A schematic representation of the model showing the overall fold and the
probable site of the large insert is presented in Fig. 8.
In order to correlate observations from kinetics with structural features
in the enzyme, we constructed models of cyprosin complexed with ligands in
the active site to mimic the transition-state analog. Most of the pockets in
cyprosin are very similar to those in the barley-grain aspartic proteinase
(Guruprasad et al. 1994); the only difference is near the S2' and S3' pockets.
This difference is due to His295 in cyprosin compared with Arg295 in the
barley enzyme.

7 Tissue-Specific Accumulation of Cyprosin


Studies on the expression of cyprosins in various organs and tissues of C.
cardunculus were carried out by measuring the proteolytic and milk-clotting
activities, as well as investigating the occurrence of the proteins and cyprosin

Cynara cardunculus sUbsp.flavescens (Cardoon)

147

mRNA in various extracts, and by immunogold-Iabeling of appropriate tissues


(Cordeiro et a1. 1994a,b).
7.1 Accumulation of Cyprosins in Flowers
The accumulation of cyprosins in flowers at several stages of development (2
to SOmm in length), styles and corollas from mature flowers as well as seeds
and leaves have been studied (Cordeiro et a1. 1994b). In early stages of
development, flowers (2-7 mm long) showed relatively low specific proteolytic
activity (20 to 30% of mature flowers). During flower development, a continuous increase in the proteolytic activity was observed. The highest activities
were obtained in mature flowers (SOmm long) and in isolated styles and
corollas from such flowers . Seeds and leaves showed no significant proteolytic
activity. The highest clotting activities were found in extracts of styles and
corollas isolated from mature flowers. No clotting was observed with extracts
made from seeds or leaves.
These results are confirmed by SDS-PAGE and immunoblots. As shown
in Fig. 9, the presence of cyprosin 3 can be detected in very early stages
(flowers of 2mm length) , showing that the enzyme is formed already at the
beginning of flower development. Cyprosins 1 and 2 are present at a later stage
(flowers of 7mm length).

E
E

E E
E E

E
E

E
E

E
E

E
E

E
E

E
E
~

.....

If)

<"J
(//

(//

(//

(/)

(//

<"J
(/)

If)

N
(//

(/)

(//

II:

:> :>

:>

:>

:> :>

:>

:>

:>

o....I

11.

11.

E
E
0

III
II:
W

III
II:

11.

11.

....I

....I

III
II:

....I

11.

.....

III
II:

....I

11.

......

III III
II: II:

~ ~

11.

....I

...

III
II:

~
....I

<"J

III
II:

11.

<"J

III
II:

0
(//

E E
E E

....I

11.

oct
....I
....I

zW 0a:

11.

....I

II.

08
..... M

(//

0
0

(//

W
....I

(//(//(/)

(//WW Ill

00:
Woctocto

~ (/)....1....1:2
www-

(//

large subunit
cyprosln 3
31 .0 kOa

large subunit
cyprosin 1 and 2

14.4 kOa

Fig. 9. Immunostained Western blot following SDS-PAGE gel of crude extracts from various
organs and tissues of Cynara cardunculus. The samples (to ~g of protein per lane) were flower
buds and flowers at various stages of development, corollas and styles from mature flowers, seeds,
leaves, and midribs. (Cordeiro et al. 1994b)

148

M.e. Cordeiro et al.

The plant-clotting enzymes are specifically expressed in the flowers of


cardoon; they have been detected only in the flower organs, and mainly in
styles and corollas. Cyprosins are not present in leaves, midrib, or seeds. The
enzymes are present in an active form in flower tissues at very early stages of
flower development (2mm) and they are accumulated in the flowers along
with flower development.
7.2 Expression of Cyprosin Genes During Flower Development

Total RNA, isolated from different organs, including several stages of flower
development, styles from mature flowers, bract, and leaf, was separated by
agarose gel electrophoresis, blotted onto a GeneScreen membrane and hybridized with the 32P-Iabeled EcoRI 1.7-kb insert of cyprols. The cDNA clone
hybridized to a 1.8-kbmRNA from flower and bract tissues, but there was no
detectable hybridization to mRNA from leaves (Cordeiro et al. 1994a).
The intensity of hybridizing transcripts increased from early stages of
floral development (flowers 6-10mm in length) to later stages of floral development (flowers up to 40mm in length), while in the later stages of floral
development (flowers 50mm in length and styles from open flowers) no hybridization signal was visualized, indicating that the synthesis of mRNA encoding the cyprosin starts in early stages of floral development and switches off
at maturation of the flower. These findings are basically in agreement with the
enzyme activity measured in extracts made from flowers at different stages of
development and also with the organ-specific expression of cyprosins.
In conclusion, the genes coding for the cyprosins are expressed during
early stages of flower development (up to 40mm long). Furthermore, the
expression of the cyprosin genes seems to be specific for organs present in
flowers and bracts.

8 Localization of Cyprosins in the Flower Tissues


The localization of cyprosins in flower tissues was performed by immunogold
labeling using cyprosin antibodies. Studies were carried out with flowers fixed
before anthesis at several stages of development.
Silver enhancement of immunogold-Iabeled flower cuts (1-2,um) allowed
the detection of specific labeling of the cyprosins by light microscopy. Figure
10 shows the localization of cyprosins in transversal cuts made at the intermediate region of flowers (35-40mm in length). The cardoon proteases appear to
be specifically located in the epidermal cell layer of styles. No other tissues
from styles and corollas have shown any labeling. The cortex, the transmitting
tissue, and vascular bundles of styles, as well as the epidermal cell layer and
cortex of corollas, showed no labeling.
Cuts obtained from the basal region of flowers show a less intense labeling
when compared to cuts from the intermediate region of the same flowers.

Cynara cardunculus subsp. fiavescens (Cardoon)

149

Fig. lOA-C. Localization of cyprosins in styles of Cynara cardunculus. Visualization of immunogold labeling by the silver enhancement technique. A Transversell section of style showing
presence of cyprosins in the epidermal cell layer. B Amplified view of cells of the epidermal layer
of styles. C Control treated with BSA, invertase and rabbit preimmune serum. C Corolla; Cu
cuticule; Ep epidermal layer; PC parenchymatous cortex; TT transmitting tissue; VB vascular
bundle. Bars (Cordeiro et al. 1994b)

150

M.e. Cordeiro et al.

These data indicate a differential distribution of cyprosins along the flower,


and a higher accumulation of the enzymes in the upper part of the flower.
Also in electron microscopy, specific labeling of cyprosin antibodies was
detected in the epidermal cell layer of styles using a gold-labeled secondary
antibody. Gold particles (10nm), concentrated in electron-dense agglomerates, are visualized in the hyaloplasm dispersed all over the cell. They are not
concentrated in plastids, mitochondria, or the nucleus. The structural preservation used in immunogold labeling techniques does not allow one to clearly
distinguish between the endoplasmic reticulum, Golgi apparatus, coated vesicles, and cytosol, because osmium tetra oxide is not used during this fixation
and, for this reason, cellular membranes are not stained.
Since cyprosins are glycoproteins and appear to contain a signal peptide,
it is unlikely that a cytosolic form exists. So, their detection in the cytosol can
be explained by cyprosins being transiently present in either the endoplasmic
reticulum, Golgi apparatus, or coated vesicles during synthesis and processing
at this stage of flower development (before anthesis). Further studies are in
progress to confirm this hypothesis.

9 Possible Physiological Functions of Cyprosins


9.1 Protection of Styles from Other Organisms

Studies on the localization of cyprosins by immunogold labeling and electron


microscopy have revealed relatively high concentrations of cyprosins mainly in
the violet part of styles, which is not protected by bracts and the capitulum
receptacle, and in the epidermal cell layer of styles. This specific localization
for these proteolytic enzymes has led to the elaboration of the hypothesis that
cyprosins might be involved in some mechanism of protection against other
organisms such as insects, viruses, or microorganisms. These enzymes have a
high proteolytic activity and their localization in the peripheral cell layer of
styles appears to be very strategic as a defence mechanism against pathogens.
9.2 Degradation of Proteins During Flower Senescence

Studies on the expression of cyprosins along floral development have revealed


the presence of cyprosins in early stages of flower development and accumulation of the enzymes in flower tissues towards later stages of flower development. Based on these observations, it may be suggested that cyprosins are
involved in the degradation of proteins that may be involved in the development and/or growth of flowers. They may also be functioning as activating
enzymes involved in one or more pathways important in such processes.
SDS-PAGE of crude extracts from fresh cardoon flowers at different
stages of development reveal a reduction in the number of protein bands in the
gel as flowers become senescent. A change in protein population is observed

Cynara cardunculus sUbsp.fiavescens (Cardoon)

151

along flower development. Similar results have been obtained by SDS-PAGE


studies of flower senescence in daylily (Hemerocallis; Lay-Yee et a1. 1992).
The inhibition of flower senescence by cycloheximide suggested that flower
senescence requires de novo synthesis of specific proteases or other proteins
which facilitate protein breakdown.
Dried flowers of C. cardunculus show the highest content of cyprosins and
the highest clotting activity. The fact that cyprosins are not degraded themselves in the flower and that they are accumulated until later stages of development and during flower senescence supports the assumption that they are
degrading other proteins during development of the flower and most particularly during flower senescence.
9.3 Control of Flower Development

The expression of cyprosin mRNA is similar to the expression of genes controlling flower development. Examples are the agamous and plenna genes
responsible for the c function during floral organ development (Cohen 1992).
The c function in floral meristem is responsible for the development of fertile
organs (stamens and carpels). The agamous and plenna mRNAs are first
detected in initial stages of organ development at a region destined to form
stamen and carpel primordia. The expression continues further and until
stages close to mature organs. Control of flower development is performed in
a synergistic and/or antagonistic interaction between genes involved at each
stage.
The expression of cyprosin mRNA has been detected at early stages
of flower development and is switched off at later stages and in mature
styles.
Based on this general knowledge about expression of genes involved in
flower development, we can assign a hypothetical role for cyprosins and
cyprosin genes in the control of flower development and organ differentiation.
They may act directly by controlling the differentiation of one specific organ,
or by preventing the accumulation of other flower gene products that could
give rise to phenotype alterations.
Acknowledgments. This work was supported by grants from the Swedish Natural Science Research Council, the Swedish Council for Forestry, and Agricultural Research to PEB.

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Cordeiro M, Guruprasad K, Blundell T, Pais MS, Brodelius PE (1994c) Rule-based comparative
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Cordeiro M, Pais MS, Brodelius P (1997) Cynarases (proteases) of Cynara cardunculus: studies on
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X Ephedra Species: In Vitro Culture,


Micropropagation, and the Production of Ephedrine
and Other Alkaloids
N.A. O'DowD l,4, P.G. MCCAULEY\ G. WILSON2, J.A.N. PARNELLl,
T.A.K. KAVANAGH 3, and D.J. MCCONNELL3

1 Introduction
Botanically, Ephedra (Fig. 1) is a member of the smallest and most problematic division of flowering plants, the Gnetopsida, and major questions remain
unanswered about the taxonomy of the Gnetopsida and the evolutionary
relationships of the different genera within the division. Ephedra is the largest
and most widely distributed genus in the Gnetopsida, a subgroup of the
gymnosperms. Many anatomical and reproductive characters of Ephedra are
angiosperm-like (Alosi and Alfieri 1972; Friedman 1990; Carlquist 1992;
Simcha 1994). Recent molecular and chemical studies support the view that
the Gnetopsida are the closest living relatives of the angiosperms but that the
angiosperms are not derived from them (Martin and Dowd 1986; Chase et al.
1993; Doyle et al. 1994; Nickrent and Soltis 1995).
Pharmacologically, Ephedra has been the main botanical source of the
active alkaloids I-ephedrine (E) and d-pseudoephedrine (PE) for thousands of
years, with records of its medicinal use dating to 5000 years B.P. (Evans 1989).
The alkaloids E and PE remain important drugs today - the current world
consumption of d-pseudoephedrine salts (PE-sulphate and PE-hydrochloride)
stands at 1000-2000 tonnes per annum with a value of approximately $100-200
million (c. O'Brien, pers. comm.).
Powdered Ephedra stems are used in traditional herbal medicines as a
hypertensive aid to treat asthma, nose and lung congestion, hay fever, and
several other ailments. E. sinica and E. equisitina are the two main species
exploited. The role of I-ephedrine and d-pseudoephedrine in modern medicine has been changing slowly as new applications for these alkaloids are
realised. Acting as potent vasoconstrictors, they can be used to elevate blood
pressure and increase heart and respiratory rate (Gilman et al. 1990). They are
very close to adrenaline in structure and pharmaceutical activity, but have the
advantage that they can be administered orally (Fig. 2). Uses for the Ephedra
alkaloids continue to expand. L-ephedrine (E) is currently under investigation

School of Botany, University of Dublin, Trinity College, Dublin 2, Ireland


School of Botany, University College Dublin, Belfield, Dublin 4, Ireland
3 Dept. of Genetics, University of Dublin, Trinity College, Dublin 2, Ireland
4 Present address: TEAGASC, Food and Agriculture Development Authority, Kinsealy
Research and Development Centre, Malahide Road, Dublin 17, Ireland
I

Biotechnology in Agriculture and Forestry, Vol. 41


Medicinal and Aromatic Plants X (ed. by y'P.S. Bajaj)
Springer-Verlag Berlin Heidelberg 1998

155

Ephedra Species

Fig. 1. An Ephedra minima plant with ripe female cones (megasporangiate strobili on which the
bracts have become swollen). Ephedra species produce one to two seeds per cone

cgf,
cY$cr
n
,
o XlQJ
",01-1

Fig. 2. Chemical structures of


the four major Ephedra
alkaloids. Adrenaline is
shown for comparison.
differing from I-ephedrine
(E) only by a couple of
hydroxyl groups on the
phenyl ring

NHCH

rn

NHCH l

I-ephedri ne

d-pseudoephedrine

I-norephedrine

d-norpseudoephedrine

HO

HO

HO

~
o X
",H

ol'

."

GI

NHCH

adrenaline
(epinephrine)

J
J

N.A. O'Dowd et al.

156

as a therapeutic agent for the treatment of obesity (Zgourides et al. 1989). Lephedrine has been linked with toxic psychosis (Whitehouse and Duncan
1987) with the result that the demand is increasing for the stereoisomer, dpseudoephedrine (PE), which has fewer side effects than I-ephedrine and a
weaker, longer-lasting effect upon the central nervous and cardiac systems. A
recent development is the introduction by Schering-Plough of a new combination product containing pseudoephedrine sulphate and Loratadine, a nonsedating antihistamine (Sanchez Garcia and Trejo Tapia 1993).
Two current methods of producing E and PE are outlined in Fig. 3. China
and Pakistan are the main producers. Attempts have been made to cultivate

fermentable sugar
(molasses)

CHO
benzaldehyde

"Saccharomyces cervisiae

'\
~III
lff-C--CH
OH 0

phenylacetylcarbinol (PAC)
L-1-phenyl-2-oxo-propanol
CH 3 NH 2
methylamine

Ephedra plant extract

I catalytic reduction

OH H

h-b-CH
IQ\
~I
I
H

NHCH 3

l-ephedrine
Qllnd

main sources
Pakistan

E. sinica
E. equisitina
E. intermedia
[Hu, 1969,

Yin, 1994
Man & Yang, 1995)

E. gerardiana
E. nebroidensis

chemical
conversion

'0'h- b-- CH
~II

OH NHCH 3

d-pseudoephedrine
Fig. 3. Current processes employed in the manufacture of I-ephedrine and d-pseudoephedrine

Ephedra Species

157

Ephedra in Australia, England, Kenya and the United States, but these ventures failed, largely for economic reasons (Morton 1977).
Although it is currently possible to manufacture these compounds from
benzaldehyde via a yeast fermentation and catalytic reduction, several species
of Ephedra continue to be exploited for these high-demand pharmaceutically
active compounds (Fig. 3). Therefore, the continued importance of these wellknown Ephedra alkaloids appears assured for some time to come, and there is
the prospect that other pharmacologically active agents may be found in
Ephedra and other members of the Gnetopsida.

2 Distribution of the Ephedrines


From the definitions outlined by Southon and Buckingham (1989), the
Ephedra alkaloids, I-ephedrine (E), nor-ephedrine (NE), d-pseudoephedrine
(PE) and nor-pseudoephedrine (NPE), can be classed as proto alkaloids (Fig.
2). This definition is supported by Hegnauer (1988), who classes alkaloids
according to their biogenetic pathways. These compounds are also referred to
as phenylalkylamines or phenylpropanoids, terms which give more information on their structure. Nevertheless, with these words of caution, we will refer
loosely to the ephedrines as alkaloids.
The literature shows that there appears to be wide variation in alkaloid
content between species (Table 1). However, several problems arise in assessing alkaloid yields of Ephedra. Firstly, the compounds I-ephedrine (E), dpseudoephedrine (PE), nor-ephedrine (NE), nor-pseudoephedrine (NPE),
methyl-ephedrine (ME), and methyl-pseudoephedrine (MPE) are difficult to
separate and identify.
Many published reports of alkaloid production by Ephedra (Table 1) have
relied upon TLC; some do not refer to the analytical method used. In fact,
TLC is not sufficiently discriminating on its own to differentiate between the
Ephedra alkaloid stereoisomers. Moreover, ethanol extracts of parent plant
and in vitro-grown tissues often contain compounds which cochromatograph
with E and PE standards and stain positively with ninhydrin, but which HPLC
analysis reveals are not phenylalkylamines (O'Dowd 1991). The reported
occurrence and levels of alkaloids in the Ephedra genus, especially in the older
references depending only on TLC, must therefore be questioned.
Analytical techniques such as HPLC, which accurately determine and
quantify these compounds, are quite recent developments (Barkan et al. 1981;
Sagara et al. 1983; Jian et al. 1988; Liang et al. 1990, 1991). It is now feasible to
survey the distribution of phenylalkylamines throughout the Ephedra genus
and to construct chemical profiles within and between species. Such analysis
would enable the critical testing of the suggestion that there is evidence of
chemical races within the species Ephedra distachya which differ in their
alkaloid composition (Evans 1989). It may be possible to distinguish between
genetic and environmental factors influencing alkaloid synthesis.

N.A. Q'Dowd et al.

158

Table 1. Reported alkaloid content of different Ephedra species and Ephedrae herbae. Alkaloid

levels given as percentage dry weight of stems. L-ephedrine (E), d-pseudoephedrine


(PE), norephedrine (NPE), -norpseudoephedrine (NPE), methylephedrine (ME),
methylpseudoephedrine (MPE)
Species

Alkaloid content

Reference

E. americana var.

0.02% E; 0.01 % PE

Q'Dowd (1991)

0.04% E; 0.13% PE; 0.01 %


NE Leeds University
E and PE present
PE present

Q'Dowd (1991)

andina
E. andina

E. alata Decne
E. alenda (Stapf)
Andreanszky
E. altissima Desf.
E. americana Humb.
& Bonpl.
E. andina Poepp.
E. californica S. Wats.
E. ciliata Fisch et Mey
E. dis tach ya"

E. dis tach ya var.


helvetica

E. equisitina Bunge

E. foliata Boiss
E. distachya
(continued)
E. fragilis Desf.
E. fragilis ssp.
camplyopoda
E. gerardiana Wall b

E and PE present
E present
E present
E and PE present
Afghanistan. No reference
to alkaloid but used locally
as antiasthma tic
E and PE both present
Total alkaloid: 0.29% in
nodes and 0.69% in
internodes. E, NE, PE, ME
and MPE present
0.07% E; 0.2% PE
0.04% E; 0.05% PE; 0.02%
NE; 0.05% NPE
0.75-1.0% E with variable PE
1.75% total alkaloid, 1.58% E
2.09% total alkaloid, 1.0% E,
0.05% NE, 0.4% PE, 0.47%
NPE, 0.05% ME, trace MPE
Mongolia. 1.72% total alkaloid
0.8% E; 0.5% PE
Iraqi species pharmacologically
active
Devoid of E
India (Rajasthan) during
different seasons - 0.01 % E
Thar Desert, India; 0.0% trace alkaloid
E and PE present
Menorca; 0.06% E; 0.03% PE;
0.05% NE; 0.09% NPE
0.14% E; 0.51 % PE
Total alkaloid - 0.8-1.4% of
which ~50% E: PE, ME,
NE, MPE, NPE also present
Rajasthan, India. 1.7% E
Japan. 0.03% E and 0.97-1.07%
PE. NE and ME not detected

Smith (1977)
Willaman and Schubert
(1961)
Willaman and Schubert
(1961)
Willaman and Schubert
(1961)
Smith (1977)
Willaman and Schubert
(1961 )
Younos et al. (1987)
Yamasaki et al. (1973)
Kasahara et al. (1986)

Q'Dowd (1991)
Q'Dowd (1991)
Windhotz et al. (1983)
Morton (1977)
Jian et al. (1988)
Gazaliev et al. (1988)
Q'Dowd (1991)
Al Sarraj et al. (1985)
Chaudhri (1957)
Yamasaki et al. (1973)
Chopra et al. (1956)
Smith (1977)
Previously unpubl.
Q'Dowd (1991)
Morton (1977); Duke
(1986)
Ramawat and Arya
(1979a)
Yamasaki et al. (1974)

Ephedra Species

159

Table 1. Continued

Species

Alkaloid content

Reference
Chaudhri (1957)

E. gerardiana vaL
sikkimensis
E. gracilis R. Phil

Total alkaloid is 1.0-2.5%,


of which 60-70% is E,
40-30% PE
0.1 % E; 0.12% PE; 0.03% NE;
0.04% NPE
Bhutan, 3600m altitude - 0.13%
E, 0.41 % PE, trace ME
E and PE present

E. helvetica CA. Mey.

Main alkaloid is E - not


quantified
E and PE present

E. intermedia'

Low in E but quite high in PE

E. helba

E. major

E. major vaL procera


E. minima
E. monosperma S.G.
Gmel.
E. monostachya L.
E. nebroidensis Tineo d
E. nebroidensis var.
procera
E. nevadensis S. Wats.
E. pachyclada Boiss.
E. procera Fisch &
Mey.
E. saxatilis
E. scoparia
E. sinica Stapf.

0.2% E, 0.04% NE, 1.16% PE,


0.15% NPE, 0.02% ME,
trace MPE 1.56% total
alkaloid
0.5%-\.5% total alkaloid
Afghanistan. 0.006% E, 0.002%
PE. Bachluchistan. 0.35% E,
0.18% PE, trace ME
0.8-1.9% total alkaloid of which
0.1-0.25% is E, 0.6-1.4% PE,
also ME, MPE, NE and NPE
0.06% E; 0.15% PE
From Wiirzburg Botanic
Gardens; Germany, 0.34% E;
0.18% PE From Marburg
Botanic Gardens; Germany
0.2% E; 0.05% PE
0.27% E; 0.75% PE
0.5% E; 1.04% PE; 0.05% NE;
0.06% NPE
E and PE present

Q'Dowd (1991)
Yamasaki et al. (1974)
Willaman and Schubert
(1961)
Akiba et al. (1979)
Willaman and Schubert
(1961)
Morton (1977);
Nishimoto (1980)
Jian et al. (1988)

Chaudhri (1957)
Yamasaki et al. (1974)

Liu et al. (1993)


Q'Dowd (1991)
Q'Dowd (1991)

Q'Dowd (1991)
Q'Dowd (1991)
Smith (1977)

E and PE present
Material from Pakistan reported
to contain 1.3 % total alkaloid
Baluchistan. 0.57-1.12% E.

Smith (1977)
Shah and Shah (1966)

Devoid of alkaloid
E and PE present
Total alkaloid ~1.4%

Duke (1986)
Smith (1977)
Pelt et al. (1967)

Devoid of alkaloid
Trace E; 0.01 % PE
1-2.5% total alkaloid
1.38% total alkaloid of which
0.77% E, 0.05% NE, 0.31 %
PE, 0.14% NPE, 0.09% ME,
0.12% MPE

Q'Dowd (1991)
Q'Dowd (1991)
Hu (1969)
]ian et al. (1988)

Khan et al. (1983)

160

N.A. O'Dowd et al.

Table 1. Continued

Species

Alkaloid content

Reference
Liu et al. (1993)

E. triandra Tul.

0.9-2.3% total alaloid of which


0.4-1.6% E, 0.2-0.8% PE,
also ME, MPE, NE and NPE
Contains E

E. tweediana c.A.
Mey
E. viridis Coville
E. vulgaris L.c. Rich.
(see E. distachya)
Chinese Ephedrae
Herba

Pakistani Ephedra
Herba
Ephedrae Herba
Taiwan market
Major ephedrine
yielders

Contains E
E and PE present
0.0% E; 0.05% PE
Contains E, PE, NE and ME
30 mixed samples over 5-year
period: 0.8-1.8% total
alkaloid
30 mixed samples over 5-year
period: 2.5 % total alkaloid
0.9-2.3% total alkaloid of which
0.4-1.6% E, 0.2-0.8% PE,
also ME, MPE, NE and NPE
E. sinica Stapf. (China)
E. equisitina Bunge. (China)
E. gerardiana Wall. (India and
Pakistan)

Willaman and Schubert


(1961)
Willaman and Schubert
(1961)
Willaman and Schubert
(1961)
O'Dowd (1991)
Smith (1977)
Takahashi et al. (1982)
Takahashi et al. (1982)
Liu et al. (1993)
British Pharmaceutical
Codex (1975)

, E. distachya is often referred to as E. vulgaris.


Synonyms of E. gerardiana Wall. are E. major Host, E. vulgaris Rich. and, until recently, E.
nebroidensis Tineo (now recognised as a separate species).
C E. intermedia also known by the synonym of E. pachyclada.
d E. nebroidensis often referred to under the synonyms; E. major Host., E. gerardiana Wall. and
E. vulgaris.

It is also recognised that there is a marked seasonal vanatlOn in the


alkaloid yields of Ephedra (Chaudhri 1957; Hu 1969; Kasahara et al. 1986).
Furthermore, by our own observation, uneven sampling of plant tissues and
the age of the tissues sampled affects the recorded alkaloid levels (O'Dowd
1991). Kasahara et al. (1985) found that the total alkaloid content in nodes of
several Ephedra species was only 40% of the concentration found in the
internodes (approximately 0.29 and 0.69% dry wt., respectively). The distribution of E and PE in different organs of E. minima was studied (O'Dowd 1991)
and the results are given in Table 2. Variation in alkaloid content also occurs
along the length of the stem with low quantities in the very young tip growth
and the basal, woodier tissues (Fig. 4, 5).
It is still not known where the site/s of synthesis of the compounds are
located in the plant and it is important to note that high levels in certain tissues
may indicate sites of accumulation rather than of production. As much of this
chapter concerns the secondary metabolism of Ephedra, we suggest Gottleib
and Kubitzki (1984) for a review of flavonoid, terpenoid and other constituents of this genus. Table 3 references several subsequent reports.

Ephedra Species

161

Table 2. Distribution of I-ephedrine and d-pseudoephedrine within a 2-year-old plant of E.


minima (After O'Dowd 1991)

Plant part

Dry weight
I-ephedrine (%)

Dry weight
d-pseudoephedrine (%)

Achlorophyllous, subterranean shoots


Top 2cm of shoots
Primary shoots (internodes)
" " (nodes + leaf scales)
Secondary shoots (internodes)
Tertiary shoots (internodes)
Lower 2cm of primary shoots
Rhizome
Main root
Fine, fibrous roots

o
o

0.17
0.04
0.16
0.05
0.02

0.16
0.06
0.17
0.04
0.02
0.01
0.01
0.01

OJn

0.01
0.01

Table 3. Compounds isolated from Ephedra subsequent to those listed in Gottleib and Kubitzki's

literature review (1984)


Species

Compound

Reference

E. sinica

Kaempferol and herb acetin (8-hydroxykaempferol), 3-methoxyherbacetin,


apigenin, tricin, apigenin-5-0rhamnoside and kaempferol-rhamnoside
Pelargonindin, apiginenidin and delphindin

Purev et al. (1988)

Mahuannin D
Catechins
Alkaloids
(- )-Ephedrine, (- )-norephedrine,
(- )-N-methylephedrine, (+ )-Wephedrine, ( + )-nor-W-ephedrine, (+ )N-methyl-W-ephedrine
Ephedralone
Lignans
( )-Syringaresinol

Kasahara and Hikino (1983)


Duke (1986; cited by)

E. andina,
E. breana and
E. frustillata,

species not given


E. gerardiana

Various - see
Refs.
E. alata
E. alata

Gurni and Wagner (1984)

Southon and Buckingham


(1989) (and Table 1)
Nawwar et al. (1985)
Nawwar et al. (1985)

Glycan~

E. distach ya
E. gerardiana
E. gerardiana
E. alata

Ephedran A B, C, D and E
Saponin
Tannins
Gallic and elJagic acids
Digalloyl-glucoside
Nilocitin

Konno et al. (1985)


Duke (1986; cited by)
Duke (1986; cited by)
Nawwar et al. (1985)

In summary, there is strong evidence that the age and type of tissue
sampled, environmental growth conditions and the method of analysis used all
strongly influence the alkaloid content measured for that species. These factors are very important to keep in mind, especially when analysing small
quantities of plant tissue, and also make it very difficult to assess much of the
older work shown in Table 1.

Fig. 4. Distribution of I-ephedrine (eph) and d-pseudoephedrine (Peph) in stems of a 2-year-old


E. minima plant. The percentage dry weight of alkaloid was calculated per internode (1 tip; 11
base). Highest levels are found in older, green internodes with no phenylalkylamines occurring in
the tips (9-11) or the woody, basal internodes (1-3)

2.1 Analytical Methods

Two methods of analysis were used to determine alkaloid presence and concentration in plant tissues; high performance liquid chromatography (HPLC)
and thin layer chromatographic analysis (TLC).
Sample Preparation. A known quantity of tissue (0.1-0.2 g parent plant or 0.51 g cultured cells) was ground in liquid nitrogen and extracted by 30-min
sonication in a known quantity (0.5-1 ml) of ethanol or 50mM Tris, pH 8.
Samples were spun and the supernatant pipetted off.
Chromatographic Conditions. Adapted from Barkan et al. (1981). HPLC was
carried out on a Spectra-Physics Model 8000 HPLC equipped with an SP 8000
integrator and Spectra Physics Model 770 UV-visible detector set at 21Onm. A
NOV A-PAK phenyl (reverse phase) column was used (IO.um particle size;
15cm X 3.9mm internal diameter). A guard column was employed. Mobile
phase was 1 % acetonitrile in 50mM monobasic sodium phosphate, flow rate
Imlmin- 1, 20.uM injection loop.
For TLC several systems were tested, including normal and reverse phase
(O'Dowd 1991). Best results were achieved with aluminium-backed 0.2-mmthick silica gel 60F254 stationary phase and butan-l-ol: acetic acid: water
(4: 1 : 1) mobile phase, localisation agent ninhydrin.
2.2 Biosynthesis of Ephedra Alkaloids

The alkaloids I-ephedrine (E), d-pseudoephedrine (PE), norephedrine (NE)


and norpseudoephedrine (NPE) (Fig. 2) are found in different proportions in
different Ephedra species (Table 1). Some of these compounds have been

163

Ephedra Species

if'

1r.>:
:1

il

il
!I

II
I)
!

!
I

:1

;. ~:.1
"."!
(""!

,: ...!

I
t

Iijj

1,.1

~.

':'!

,
~

"I

rt' :,
~

"

"1

,I

,:,.1
I ..

f:!

,,f' Lf")
'

.:-.. j
.J'

(\1

Fig. SA-Co HPLC chromatograms of E. minima stem methanol extracts. A Top 2 em of shoots
contain no alkaloid, B Tertiary stems contain small quantities ofE (0.04% dry wt.) and PE (0.05%
dry wt.), C Primary stems contain highest levels with 0,18 and 0.19% dry wt. E and PE, respectively. Retention times vary between samples, Confirmation by spiking with E and PE standards
and TLC

found in other plant species as well, for example E and/or PE have been found
in Sida cordi/olia (Ghosal et al. 1975), Catha edulis (Kalix 1991), Roemeria
re/racta (Southon and Buckingham 1989), Pinella ternata tubers (Oshio et al.
1978), Taxus baccata, Aconitum napellus (Duke 1986) and Hamelia patens

N.A. O'Dowd et al.

164

(Chaudhri and Thakur 1991). The fact that they appear in such a broad
spectrum of plants suggests that they could be derived from the same primary
metabolite, e.g. amino acid or the same breakdown product of a primary
metabolic pathway, but, in fact, little has been known until recently about the
biosynthesis of the ephedrines and it is not known if these compounds are
synthesised by the same biosynthetic route in the different genera. Table 4 lists
synonyms commonly used in reference to this group of compounds. It has not
been easy to establish the biosynthetic pathway for the ephedrines (Fig. 6).
Efforts have been directed at finding the main sources of the C6 ring, the C3
side chain, the amino group and the N-methyl group.
Phenylalanine was an obvious potential precursor - it is a C6-C3 compound
with an amino group on the C2 carbon of the C3 side chain, as in the ephedrines,
but in some ways this similarity may have been misleading certainly, it has been
established that in Ephedra and Catha edulis, label from [14C] phenylalanine
can be found in I-ephedrine (Shibata and Imaseki 1956; Yamasaki et al. 1973;
Cordell 1981, Grue-Sji'lrensen and Spenser 1988, 1989). Phenylalanine was
reported to be an efficient precursor of d-norpseudoephedrine (cathine) in the
latter species (Shibata and Imaseki 1953). Therefore, it seems clear that the
ring can be derived from phenylalanine, although it cannot be assumed to be
the only source. In Ephedra, phenylalanine was found only to provide a C6-C1
unit (Yamasaki et al. 1973) and not the complete aminophenylpropanoid
system of E as originally thought.
Phenalanine ammonia lyase (PAL) catalyses the conversion of 1phenylalanine to trans-cinnamic acid by elimination of ammonia. Cinnamic
acid, in turn, can be hydrolysed by f3-oxidation into benzoic acid and acetic
acid. Although neither of these steps has been demonstrated in Ephedra, they
are generally common within the higher plant kingdom and are assumed to be
present in the Ephedra genus. Feeding experiments using [7- 14 C]-benzoic acid
confirmed that benzoic acid was incorporated with high efficiency into E in
Ephedra (Yamasaki et al. 1973). Since the incorporation of [14C]-benzoic acid
was much higher than that of p4C]-phenylalanine, Yamasaki et al. (1973)
suggested that benzoic acid may be derived by an alternative route, for example through shikimic acid. Therefore, benzoic acid could provide a C6-C 1 unit
which is subsequently condensed with a C2 unit to form the carbon backbone
ofNE/E.
Table 4. Names and synonyms of main ephedrine alkaloids

Common name
Ephedrine
Pseudoephedrine
Norephedrine
Norpseudoephedrine
Cathinone

Synonyms
(lR,2S)-( - )-ephedrine
(lS,2S)-( +)-pseudoephedrine
(lR,2S)-( - )-norephedrine
(lS,2S)-( + )-norpseudoephedrine
cathine
S,S( + ) phenylpropanolamine
(S)-( - )-2-amino-l-phenylpropanone-1one
S( - )alpha-aminopropiophenone

Ephedra Species

165

~CH2-TH-COOH
~j
NH3
t
~

PHENYLALANINE

NH2
phenylalanine ammonia lyase

@- ~-C-CH
II

PHENYLPROPANEDIONE
3

.. ' ... amino group from another amino acid

@-~-TH-CHJ
.

reductlOn

CATIllNONE

~H2 NADH+H+/NADPH+W
NAD+ / NADP

(cofactors

?)

OH

NOREPHEDRINE
OR

CH -CH-CH 3

S-adenosylmethionine
methylation

@-~:-TH-CHJ
NHCH 3

EPHEDRINE
OR
PSEUDOEPHEDRINE

Fig. 6. Proposed biosynthetic pathway for the Ephedra alkaloids, I-ephedrine and dpseudoephedrine. (After Grue-Sorenson and Spenser 1993)

N.A. O'Dowd et al.

166

The identity of the Cz unit which condensed with benzoic acid to complete
the three-carbon chain remained unknown until the discovery by GrueS~rensen and Spenser (1988) that it was provided by pyruvic acid. Thus, the
carbon skeleton of the Ephedra alkaloids is generated from two fragments, a
C6-C1unit from benzoic acid and a CH3CO moiety from pyruvate. The labelled
nitrogen from [15 N]-phenylalanine was reported to be incorporated into E
(Shibata and Imaseki 1953). The transfer of 15N from phenylalanine would
appear to occur either by direct transamination from phenylalanine or another
amino acid (Fig. 6) to the intermediate precursor of NE/NPE of from free
rSN]-ammonia liberated by the action of PAL. The methyl group on the
nitrogen is derived from active methionine (Shibata et al. 1958).
Efforts to establish other biosynthetic intermediates between benzoic
acid and the ephedrines have not been successful until quite recently.
Aminoacetophenone, an intermediate suggested by Yamasaki et al. (1973),
was found not to be directly incorporated into E. An alternative structure, 1hydroxy-1-phenylpropan-2-one proposed by Grue-S~rensen and Spenser
(1988) was neither isolated nor demonstrated to provide whole or partial
labelling of E from [14C]-labelled samples of this compound. Grue-S~rensen
and Spenser (1993), working with growing stems of mature E. gerardiana
plants, have recently reported that 1-phenylpropane-1,2-dione was incorporated into the alkaloids, and claim this structure to be a hitherto unknown and
critical intermediate in the biosynthetic pathway. This compound first undergoes transamination at the Cz carboxyl group to produce cathinone. Reduction
of cathinone at C1 leads to the production of NE or NPE, which are subsequently methylated to give E/PE (Grue-S~rensen and Spenser 1993). Thus the
biosynthesis of E/PE is envisaged by Grue-S~rensen and Spenser to follow the
Table 5. L-ephedrine and d-pseudoephedrine content (% dry weight) of parent plant stem, callus
and suspension cultures of several Ephedra species (O'Dowd 1991). All cultures were derived

from stem explants and maintained on MS basal medium supplemented with 5,uM auxin and
0.25,uM kinetin
Auxin

Species
E. andina

E. equisitina
E. intermedia
E. major var.
procera
E. minima
E. fragilis ssp.
camplyopoda

Parent plant stem


Callus
Suspension culture
Parent plant stem
Callus
Suspension culture
Parent plant stem
Callus
Parent plant stem
Callus
Suspension culture
Parent plant stem
Callus
Parent plant stem
Callus
Suspension culture

2,4-D
2,4-D
2,4-D
NAA
2,4-D
NAA

Dry wt.
I-ephedrine (%)

Dry wt.
d-pseudoephedrine (%)

Trace
Trace
0.00
0.80
Trace
Trace
0.06
Trace
0.27
Trace-0.005
0.00-0.004
0.50
Trace
0.14
Trace
0.00

0.13
0.08
0.00
0.50
0.01
Trace
0.15
Trace
0.75
Trace-0.04
Trace-0.14
1.04
0.003
0.51
0.01-0.06
Trace-O.01

Ephedra Species

167

pathway outlined in Fig. 6. This pathway may not be complete, since the
condensation of the pyruvic acid and benzoic acid to produce 1phenylpropane-1,2-dione appears to be a complex reaction. It will be extremely interesting to identify the enzyme( s) and cofactor( s) involved. Further
evidence to support this pathway comes from reports that cathinone is believed to be enzymatically reduced to cathine (NPE) and NE in Catha edulis
(Kalix 1991).
In our studies, [14C)-benzoic acid was incorporated into E and PE in whole
plants of E. minima. Suspension cultures of several Ephedra species failed to
produce E and PE or synthesised only trace amounts of these alkaloids (Table
5). However, in these cultures r4C)-phenylalanine and r 4C)-benzoic acid were
incorporated into compounds (also found in parent plants) which themselves
were metabolised further but could not be isolated in sufficient quantity for
structural analysis. In a recent report Song et al. (1994a) isolated and identified
a benzoic acid derivative (6-methyl-1-heptene-5-yl-4' -benzoic acid) and a
number of p-coumaroylamino acids (Song et al. 1994b, 1995) from yeastelicited E. distachya cultures, but these are unlikely candidates for
biosynthetic precursor to NE/E.

3 In Vitro Culture Studies


3.1 Callus/Suspension Culture and Alkaloid Production

There is evidence to suggest that higher-yielding plants within a species give


rise to higher-yielding cell cultures, e.g., Nicotiana tabacum (Miller et al. 1983),
but this is not always the case (Roller 1978; Berlin 1988). Since screening at the
plant level would not be justifiable if differences in plant yields were due to
physiological rather than genetic factors, we initiated cultures from a large
number of species. Such a strategy was to allow for the eventuality that high
alkaloid-yielding species might not be amenable to in vitro culture or that
lower alkaloid-yielding intact plants might have a greater ability to
biosynthesise alkaloids in vitro.
Our studies showed that the phenylalkylamine content of plants varied
greatly between species (O'Dowd 1991; O'Dowd et al. 1993). Several types
of variation were observed. Firstly, the total alkaloid yield, expressed as
the percentage dry weight, ranged from 1.65% dry wt. for E. minima to zero in
E. saxatilis (Table 1). A further source of variation between species was
the alkaloid composition of the plants. Not all species produce the full suite
of Ephedra alkaloids (E, PE, NE, NPE, ME and MPE). Generally,
the phenyl alkylamine composition of the different Ephedra species recorded during this study agreed with previous reports (Table 1) although the
actual yields in the present study tend to be lower. Thus callus cultures were
analysed.

168

N.A. O'Dowd et al.

3.1.1 Callus Culture Growth Studies

Straus and Gerding (1963), and Konar (1963) were the first to report in vitro
culture of Ephedra. The former group isolated and maintained callus tissues of
an unidentified Ephedra species on modified White's medium (White 1963)
supplemented with 20% coconut milk and 9.uM 2,4-D. Exogenous auxin (2,4D or NAA) was required for callus growth but the auxin IAA failed to support
growth, either alone or in conjunction with a cytokinin, an observation supported in later studies (Ramawat and Arya 1976; O'Dowd et al. 1993).
O'Dowd et al. (1993) found that IBA was similarly ineffective in the initiation
of callus from stem explants of nine species of Ephedra but that it could be
used to maintain established cultures when substituted for 2,4-D or NAA. It
also proved to be the only auxin capable of supporting healthy, friable E.
minima Hao callus growth without inducing morphogenesis. It may be that
this species is more auxin-sensitive or contains higher endogenous auxin levels
than other species examined.
General conclusions concerning the in vitro growth characteristics of the
genus are difficult to draw from the literature, as the source explants vary and
the media used were often not fully defined, frequently being supplemented
with coconut milk. Table 6 summarises the in vitro culture of Ephedra by other
workers. Unless otherwise stated, Murashige and Skoog (1962) basal MS
medium was used in all studies mentioned.
One of the most -studied Ephedra species in vitro is E. Joliata, commonly
occurring in India and containing trace or no alkaloids (Table 1). Sankhla et al.
(1967a,b) examined the development of callus from mature embryos and the
subsequent production of "embryo-like structures" in some cultures. Further
work on E. Joliata is described in Section 3.5.
Production of callus from several other Ephedra species has been
achieved using stem explants as the source material for culture initiation.
Ramawat and Arya (1977, 1979c,d) examined medium composition and its
effect on callus growth of E. Joliata and E. gerardiana, a high alkaloid-yielding
species (Table 1). They tested the growth and total carbohydrate content of
callus on a range of sugars (1-6%) and found that sucrose gave highest growth
rates followed by glucose, maltose and fructose (Ramawat and Arya 1977).
Arabinose, lactose, mannitol, sorbose, soluble starch and xylose failed to
support growth. E. Joliata grew better on all treatments than E. gerardiana.
Following this, they examined callus growth and protein content on different
nitrogen sources (Ramawat and Arya 1979d). A combination of nitrate and
ammonium salts was necessary for growth (optimally 4: 1 at 400mg NI- I ). E.
gerardiana contained almost four times as much protein as E. Joliata callus on
all treatments.
Of the 18 species, subspecies or varieties of Ephedra examined by our
group, it was possible to initiate callus cultures from all of them with varying
degrees of success (O'Dowd 1991). E. altissima, E. andina, E. americana var
andina, E. distachya, E. distachya ssp helvetica, E. equisitina, E. Jragilis, E.
Jragilis var camplyopoda, E. gerardiana, E. gerardiana var. sikkimensis, E.
intermedia, E. major, E. major ssp. procera, E. minima, E. saxatilis, E. scoparia,

2. Seeds

1. Stem internodes

5. Callus from (4a)


above
Callus from (1)
above
Callus from (4b)
above

4. Stem internodes

3. Female
gametophyte

embryos
2. Pollen

5-10 /lM BAP + 2.5-5/lM


2,4-0
1O-20,uM BAP

Modified White's (WBM)


+20% CM, 7.uM 2,4-0
Modified Reinert's, 15%
CM, 7.uM 2,4-0
a) 9/lM 2,4-0 or 21.uM NAA
b) 9/lM 2,4-0 + 9,uM kin
or 36.uM BAP
c) 19/1M kin + 2.3/1M 2,4-0
gave shoot buds. 9/lM 2,4-0
+2.3/lM kin gave roots
a) 0.5-2.3.uM kin + 54/lM NAA
b) 4.5/lM 2,4-0
0.5-2.3.uM kin + 0.05-5IlM
IBA or NAA
WBM + 20% CM, 4.5/lM
2,4-0,4.7.uM kin
Liquid medium + 0.5.uM
2,4-0

I. Near-mature

E. foliata

E. fragilis

0.25 ,uM kin + 5/lM 2,4-0


orNAA

I. Stem internode

E. distachya,
E. equisitina

2. Callus from (1)


above

0.25/lM kin + 5/lM 2,4-0


orNAA
0.25/lM kin + 5/lM 2,4-0
or NAA (liquid)

1. Stem internode

E. andina

Medium'

Explant

Species

Table 6. Summary of in vitro culture studies on Ephedra

Adventitious
buds
Adventitious
buds

Callus
Callus
Shoot
regeneration
"Embryo-like
structures ..
Suspension
culture

Haploid
plantlets

Callus from
root
Haploid tissue
mass
Callus and roots
Shoot buds

Callus

Suspension
culture

Callus

Result

O'Oowd and Richardson (1993b)

O'Oowd and Richardson (1993b)

Khanna and Uddin (1976);


Uddin (1977)

Ramawat and Arya (1976)


Uddin (1977)
Arya and Ramawat (1988)
(unpubl.)
Sankhla et al. (1967b)

Bhatnagar and Singh (1984)

Singh et al. (1981)


Singh et al. (1981)

Konar (1963)

Sankhla et al. (1967a)

O'Oowd et al (1993)

O'Oowd et al (1993)

O'Oowd et al. (1993)

Reference

a,
'D

~.

(t)

"0

C/J

<:l

I'l..

~
;:,-

lO,uM BAP + 5,uM 2,4-D

1. Stem internodes

2. Callus from (1)


above

0.25,uM kin + 5,uM 2,4-D


orNAA
0.25,uM kin + 5,uM 2,4-D
or NAA (liquid)

1. Stem internode

Adventitious
buds

Suspension
culture

Callus

O'Dowd and Richardson (1993b)


Ramawat and Arya (1976)

Callus
Shoot
regeneration
Shoot
regeneration

O'Dowd (1991)

O'Dowd et al. (1993)

O'Dowd et al. (1993)

O'Dowd and Richardson, (1993b)

O'Dowd et al. (1993)

Callus

Plant growth regulator-free

Ramawat and Arya (1976)

Callus

a) 2.3,uM kin + 54,uM NAA or


0.5-4.7 ,uM kin + 4.7-107,uM
NAA
b) 0.25,uM kin + 5.0,uM NAA
or 2,4-D
c) 0.05,uM NAA + O.4,uM kin
4.5,uM kin or BAP

Reference

Result

Medium a

All media are Murashige and Skoog unless otherwise stated.

E. intermedia,
E. major ssp.
procera, E.
saxatilisa
E. saxatilis

1. Stem internodes

E. gerardiana

2. Callus from (Ia)


above
Callus from (lc)
above

Explant

Species

Table 6. Continued

f-'

-.J

0-

::;

;>o
d
o

:z

Ephedra Species

171

E. sinica and E. viridis were initiated into culture. Also two unidentified
species from Kew and Marburg University Botanic Gardens. Species producing large quantities of phenolics, especially E. andina, E. distachya and E.
gerardiana var. sikkimensis required movement to fresh medium every 24-48h
until darkening of the medium ceased. Callus growth rates were recorded on
MS medium supplemented with 1.2.uM kin and 27.uM NAA or 2,4-D. Growth
rates and callus quality varied with species. Over 14-day subculture periods,
the highest relative growth rate (Singer 1986) of 0.22 0.01 gig/day was
recorded for E. major ssp. procera (O'Dowd 1991).

3.1.2 Callus Culture Alkaloid Production

Ramawat and Arya (1979a) initiated and maintained callus cultures of E.


foliata and E. gerardiana on MS medium supplemented with 2.3.uM kin and
54.uM NAA. Eight weeks after subculture, the E. foliata callus contained no
alkaloid, while that of E. gerardiana contained 0.17% ephedrine. This yield
was one tenth that of the parent plant stems from which the callus derived. No
alkaloid was traceable within the first 6 weeks of culture, only appearing in the
later stages of growth. This may signify a switch from primary to secondary
metabolism as the nutrients in the medium become exhausted. Ramawat and
Arya (1979b) further examined ephedrine production from E. gerardiana and
E. foliata callus, looking at the influence of different plant growth regulators.
E. gerardiana callus grown on 2.3.uM kin and 54.uM NAA yielded 0.17%
ephedrine. The addition of 22.uM 2,4-D reduced the yield to 0.13%. Replacing
NAA with lEA produced the highest ephedrine level (0.3% dry weight). We
were not able to reproduce their results with E. gerardiana or any of our other
species.
By supplementing E. gerardiana callus growth medium with various
amino acids, Ramawat and Arya (1979c) reported that they were able to
manipulate the ephedrine yield. Using HPLC assays (Ramawat and Arya used
only TLC), we were unable to reproduce these results even with cultures
which produced alkaloid.
In a briefly described attempt to induce Ephedra foliata callus to produce
elevated quantities of alkaloid, Shukla (1980) subjected cultures to light of
different wavelengths. The percentage dry weight of E and PE in white-light
grown tissues was 0.1 %, increasing threefold in blue or red light. These levels
are well in excess of those in the parent plant of this species. Previous workers,
including Ramawat and Arya (1979a), whose protocol was used, did not detect
ephedrine in E. foliata cultures. The method of analysis of these tissues was
not reported, so we must consider that ephedrine was not the compound
detected. Again, the importance of using at least two different methods of
analysis to confirm the presence of these alkaloids cannot be overstressed. In
a pilot study we examined 2-month-old E. major var. procera callus and found
0.01,0.02 and 0.03% dry weight PE for white, blue and red-light-grown tissues
(O'Dowd 1991). These are the results of five pooled samples per treatment, so
it is not possible to conclude whether these results are statistically significant.

172

N.A. Q'Dowd et al.

Callus cultures from seven of our Ephedra species yielded E and/or PE but
these quantities were low compared to the parent plant (Table 5) and diminished to zero with successive subcultures over 3-5 months (O'Dowd et al.
1993). It is possible that alkaloid present in callus tissues in the early stages of
the culture could be residual from the parent plant and that we were looking
at a dilution effect over successive subcultures. Nevertheless, we did record
several cases in which callus showed steady alkaloid levels over two to three
subcultures or an increase in alkaloid level compared to the previous subculture (unpubl.), but this production could not be sustained or repeated. It is
possible that some cultures possessed the ability to produce alkaloid but the
growth conditions were not conducive. We examined heat and cold shock,
light quality and the abiotic "elicitor" vanadyl sulphate, but could not claim
convincing evidence of net synthesis of E, PE, NE or NPE (unpubl.).
3.1.3 Cell Suspension Culture Growth Studies

Strategies for the production of plant natural products using cell suspension
cultures have been widely discussed since they offer the potential for commercial exploitation. The first reports of the establishment of cell suspension
cultures of E. foliata were by Khanna and Uddin (1976) and Uddin (1977).
However, there are no descriptions of the morphology, growth kinetics or
alkaloid production of Ephedra cell suspensions apart from O'Dowd (1991)
and O'Dowd et al. (1993).
Suspension cultures have been established from a range of different
Ephedra species (Khanna and Uddin 1976; Uddin 1977; O'Dowd 1991,
O'Dowd et al. 1993). In all cases, the medium used was MS supplemented with
either an auxin (2,4-D) or an auxin (2,4-D or NAA) and cytokinin (kin). The
procedures used for formation of a suspension culture are relatively routine. A
period of growth on solid (agar) medium followed by selection of a friable
sector of callus as starter inoculum in liquid culture. In a comprehensive survey
of 12 Ephedra species, O'Dowd (1991) found that friable callus could be
encouraged by more frequent subculture prior to inoculation into liquid medium. For the initial formation of suspension cultures, a relatively high
inoculum density was preferable (up to 140g1- 1) since it was found that below
60 gl-l viable cultures could not be obtained. After inoculation (using 50ml
medium in 250ml Erlenmeyer flasks), the lumps of callus were broken apart
with a sterile spatula. Most of the remaining lumps either separated during
culture on an orbital shaker (120rpm) or were removed later by filtration.
Cultures which survived were subcultured three to four times at a high
inoculum density (80g1- 1) which was later reduced to 50gl- 1 for routine culture. All cultures were maintained at 24 1C; 16-h day at 60 1O,umolm2
(PAR).
It is clear that some species are more amenable than others to growth in
suspension culture (Table 7). Whereas callus from E. andina, E. fragilis var.
camplyopoda, E. intermedia, E. major ssp. procera and E. saxatilis callus
readily adapted to growth in liquid medium, E. fragilis, E. sinica, E. distachya,

Ephedra Species

173

Table 7. A comparison of performance of different Ephedra species established as suspension


cultures (after O'Dowd 1991). Suspensions were cultured in the same medium as that used for the
parent callus (MS with O.25,uM kinetin and 5,uM auxin)
Ephedra sp.

No. of
cultures
initiated

No. of
cultures
surviving
1 st passage

Average
duration of
survlvmg
cultures

Culture appearance

Auxin used
(5,uM)

E. andina

32

2:50
passages

2,4-D

E. distachya

3 passages

Different lines yellow


to green. Some
filamentous, others
with aggregates
(:55 mm diameter)
Cultures initially pale
green but turning
orange/brown and
dying

E. equisitina
E. tragilis

E. tragilis var.
camplyopoda

12

4 passages

E. gerardiana

3 passages

E. intermedia

14

2:14
passages

E. major
ssp. procera

2:12
passages

E. minima

2 passages

E. saxatilis

2:6
passages

E. sinica
E. sp.
(Cyprus)

4
3

0
2

4 passages

1 passage

Green, vigorous
cultures with large
aggregates (:55 mm
diameter). Often
die on sub-culturing
Cultures gradually turn
pale brown and die
Vigorous, medium
green culture, large
aggregates (:55 mm
diameter)
Yellow to pale green
with large aggregates
(:54mm diameter)
Fine, pale green culture
becoming oatmealcoloured before dying
Fine, medium green
culture
Cultures rapidly
becoming brown and
dying

NAA

NAA
NAA and
2,4-D
NAA and
2,4-D

2,4-D
NAA

NAA
NAA,2,4D and
IAA
2,4-D
2,4-D
NAA and
2,4-D

E. equisitina, E. gerardinana and E. minima survived less than four passages.

In general, suspension cultures established from green callus had a greater rate
of survival. In callus culture E. fragilis grew almost as well as E. saxatilis, with
similar growth rates, colour and friability (O'Dowd et al. 1993), yet E. fragilis
could not be established in suspension culture. Therefore there appears to be
no clear relationship between growth performance in callus culture and adaptation to suspension culture.
As can readily be seen from Table 7, there is considerable variation in
culture morphology, such as in colour, cell aggregation and cell shape, be-

174

N.A. O'Dowd et al.

tween suspensions derived from different species. E. gerardiana, E. minima


and E. saxatilis formed relatively fine suspensions with few aggregates greater
than 50 cells. In contrast, E. andina, E. tragilis subsp. camplyopoda, E.
intermedia and E. major ssp. procera formed cell aggregates up to 4 or 5 mm in
diameter, some of which contained small numbers of differentiated xylem

Fig.7A,B. Suspension cultured cells of Ephedra andina showing filmentous growth. A Characteristic branching of filaments. B Up to 1% of cells in aggregated cultures demonstrated sclariform
or spiral secondary thickening (Toluidine Blue O-stained)

Ephedra Species

175

vessels. It therefore appears that greater cell aggregation was found in the
faster-growing suspensions.
At the cellular level, Ephedra cell suspensions show variations in cell
shape. Two distinct morphologies were observed, those cultures consisting
predominantly of tangled, occasionally branching, filaments of elongated cells
(Fig. 7) and those consisting mainly of clustered spherical cells. Interestingly,
these characteristics were not species-specific; although cultures derived from
E. saxatilis and E. minima were the cell cluster type, both culture types were
found within the nine cell lines derived from E. andina. The origins and
stability of this culture variation can only be speculated upon at present but
presumably relate to some preexisting variation in the original explant or
in the selection of callus from which the suspension was derived. In the
filamentous form of Ephedra suspension cultures, the filaments became so
entangled that, at the end of the growth cycle, the cells could be picked up en
masse with a pair of forceps (O'Dowd 1991). This growth characteristic raised
difficulties for assessing culture biomass by cell counting because the cells
could not be separated satisfactorily.
Batch cultures of cell suspensions of E. andina were cultivated over a 21day growth cycle. Inoculated initially at 20g 1-1 fresh wt. (1.3 gl-1 dry wt.), the
cultures grew approximately tenfold to attain a final biomass of approx. 190282g1- 1 fresh wt. (10g1- 1 dry wt.) depending on the cell line (Table 8). The
growth rate was cell line-dependent. Uddin (1977), using a medium without a
cytokinin, reported a maximum growth index (final fresh wt. of tissue minus
initial fresh wt.) of 8 after a period of 6 weeks for E. [oliata. This growth rate
is substantially slower than those obtained by O'Dowd (1991).
Some growth kinetics of cell line E. andina (S31) over a 19-day growth
cycle are shown in Fig. 8A. The increase in both fresh weight and dry weight
follows a typical S-shaped growth curve, although there is no clear period of
constant exponential growth. An initial induction phase of about 2-3 days is
followed by a period of growth of about 7 days. The cells then enter a stationary phase about 9-10 days after inoculation. This interpretation is reinforced
by the data on mitotic index (Fig. 8B) which show a marked increase from 1 to
3.5-4% during the growth phase, subsequently declining sharply to 0.5% or
less by day 13. Cell viability (assessed by staining with fluorescein diacetate) is
uniformly high, 85-90% up to the end of the growth phase at about day 9 (Fig.
8C). However, during the stationary phase viability declines quite steeply to

Table 8. Comparison of growth rate and maximum biomass in


three cell lines of Ephedra andina. (After Q'Dowd 1991)
E. andina cell line

Mean doubling time


during growth phase
(days)

Maximum biomass
(Fresh wt. gl-l)

S 31
S 21
S 10

2.5
4.6
6.0

190 10
282 10
190 20

Cultue medium MS + 0.25,uM kin and 5,uM 2,4-D.

176

N.A. O'Dowd et al.


200

....,

,-.

12

180
160

eil
'-'

140

.,.;

10

,-.

'-'

120

..c

.::'"

-----

80

Q,j

....=
\.0

:;

60
40

eil

.,.;

100

Q,j

....,

20
0
0

10

....

\.0

'CI

4
Culture fresh wt.

Q,j

....=
:;
\.0

2 U

Culture dry wt.

0
12

14

16

18

20

Time (days)
12

B
10

....

,-.

..!.

4
,-.

8
3

eil

'-'

.,.;

:=

"'"'

....
\.0

Q,j

'CI

2 .~
....

....0

~
2
0
2

10

12

14

16

18

20

Time (days)
Fig. SA,B. Growth of E. andina cell suspension cultures grown in batch culture over 20 days. A
Culture fresh wt. and dry wt. (gl-l). B Mitotic index (%). C Cell viability (%). D Medium pH

30-35%, which may reduce the vigour of subsequent growth following


reinoculation in fresh medium if the inoculum is taken much later than day 19.
Although no studies have yet been made on nutrient uptake, it is to be
assumed that these growth patterns are related to declining nutrient concentrations and the establishment of a growth-limiting nutrient. The changes in
the medium pH values may reflect some of these changes (Fig. 8D).
The growth of cell suspensions of E. andina can be maintained over
relatively long culture periods using a form of semicontinuous culture
(O'Dowd 1991) based on a "drain and refill' dilution technique (Fidgeon and
Wilson 1987). Cultures were grown in a 1.8-1 bioreactor (Fig. 9) stirred by air
bubbling (2lmin-l). The bioreactor was calibrated for culture volume and

177

Ephedra Species
12

100

---...... 10
~

...
,.>.

90
80
70

60

",.
...=
:;

50
40

Qj

----

--.--

2
0
0

30

--~
>.

::=

:E
.;:<:\I
4l

20 U

Culture dry wt. (g.ll)

10

Cell viability (%)

0
10

12

14

16

18

20

Time (days)
6

12

0
10

::c

---.

...

Q.

:e=

Qj

,.
...:;=
Qj

----e--

--.--

~
~

,.>.

p-l

Culture dry wt.

0
0

10

12

14

16

18

20

Time (days)
Fig. 8C,D. Continued

known volumes of medium could be added and samples taken. By dilutions at


intervals approximately equivalent to the doubling time of the cells, it was
possible to maintain the fresh weight density between 7 and 14g1- 1 Using this
culture system it was possible to maintain a constant exponential growth rate
over 42 days, equivalent to cumulative total growth of 14 generations. This is
shown in Fig. 10 with, for comparison', the growth achieved during a single
growth cycle in a (19-day) batch culture. The growth rate in semicontinuous
culture was equivalent to a doubling time of 70 h (2.9 days), comparable with
that obtained during the growth phase in batch culture. These results suggest
that Ephedra cells are sufficiently shear-resistant to grow well in an air lift
bioreactor (Scragg and Fowler 1986). However, at high cell densities, mixing
problems arose. A crust of cells formed at the culture surface (sometimes

N.A. Q'Dowd et al.

178
Medium Inlet

Fig. 9. Bioreactor (1.81) used for the culture


of E. andina cell suspension cultures. (After
Q'Dowd 1991)

Filter
sterilised
air

Line
bubbled
through
2'10 "Savlon n
solution

'"c

---.-

Batch culture

-<>--

Semicontinuous culture

;:
eu

""C

:z

10

12

14

16

18

20

22

Time (days)
Fig. 10. Growth rate of E. andina (cell line S23) in batch culture and semicontinuous culture in an
air-lift bubble reactor

referred to as meringue) which, when returned to the culture, remained as a


large clump, becoming necrotic and impeding culture mixing (Scragg and
Fowler 1986). Secondly, the growing cells in the medium formed large, hollow,
spherical aggregates up to 1 cm in diameter (Fig. 11). The formation of these
was linked to the entanglement of branched filaments and the rolling agitation
of the mixing pattern. Although such biophysical characteristics may raise

Ephedra Species

179

Fig. 11. Aggregates of E. andina cells which


developed in a suspension culture grown in
an air-lift bubble reactor. Aggregates are
hollow and 5-20 mm in diameter

problems with mixing, overall, these bioreactor studies showed that it was
possible to grow a relatively large and metabolically uniform biomass of cell
suspension suitable for precursor feeding experiments.
3.1.4 Cell Suspension Culture Alkaloid Production

By comparison with intact plant tissues, levels of ephedrine and pseudophedrine were relatively low in callus and suspension cultures (Table 5).
Ephedrine and psuedoephedrine levels in suspension cultures decreased over
time and were no longer detectable after four to six passages (15-20 days per
passage). Immobilisation of E. andina cell suspensions in sodium alginate
beads did not lead to slowing of growth rate or to alkaloid production. In fact,
growth was slightly accelerated and the supernatant yielded a fine suspension
of single cells (unpubl.).
There has been little investigation of the effects of different plant growth
regulators or other culture conditions on ephedrine metabolism in cell cultures. Optimising the concentration of the components in a medium is generally an effective way to improve the production of secondary metabolites as
well as to select a highly productive cell line (Fujita 1988). An alternative
explanation for the lack of secondary metabolite accumulation in Ephedra
cultures could be due to the fact that the tissues were cultured as

180

N.A. O'Dowd et al.

undifferentiated callus or cell suspensions. This approach may fail to elicit the
complex and highly regulated patterns of gene expression found in intact
tissues and may have resulted in cultures that were neither genetically, biochemically nor physiologically competent for high-level synthesis of secondary
metabolites.
3.2 Infection of Ephedra with Agrobacterium rhizogenes

Traces of ephedrine occur in the roots of some Ephedra species in plant a


(Table 2). This distribution offers the possibility that in vitro-cultured roots
may be a potential site of alkaloid synthesis.
We made preliminary investigations of the potential for increasing both
biomass and ephedrine production by A. rhizogenes-transformed Ephedra
tissues. In total, the capacity for tumour and root production was investigated
for seven Ephedra species (E. andina, E. distachya, E. gerardiana, E. minima,
E. equisitina, E. fragilis and E. saxatilis) using the A. rhizogenes strain
Ar LBA9402 and wild-type strains Ar1000, Ar2628, Ar2629 and Ar2655
(O'Dowd 1991; O'Dowd and Richardson 1993c). Inoculations were performed
on the stems of 1- and 3-year-old potted plants and on sterile stem explants
cultured in vitro. None of the A. rhizogenes strains produced roots or tumours
on E. andina or E. distachya, but tumours and/or roots were produced from all
of the remaining species (Table 9, Fig. 12).
When excised from in vitro stem explants, tumorous tissue continued to
grow vigorously in culture in the absence of growth regulators without deterioTable 9. Percentage of wound sites on transversely cut stem explants of different Ephedra species
showing tumour and/or root development following inoculation in vitro with Agrobacterium
rhizogenes strains Ar2629 or LBA9402

Ephedra sp.

Wound sites producing


tumours (%)

Wound sites producing


roots (%)

E. andina
E. distachya
E. equisitina'

No reaction
No reaction
85%

E. tragi/is

50%. Compact, green; ~40%


becoming orange and friable

E. gerardiana'

100%. Compact, green; becoming


yellow and friable.
No reaction

No reaction
No reaction
85%. Produced from tumours
10%. Produced directly from
wound site. 4-15 roots per site
or tumour
21 %. Produced from tumours;
1-2 roots per site. Some roots
up to 4cm long
13%. Produced from tumours

E. gerardiana var
sikkimensis
E. minima

E. minima hybrid

40%. Tumours pale yellow, and


friable becoming orange.
90%. Tumours yellow and friable

Minimum of 100 wound sites per treatment.


Only LBA9402 tested.

No reaction
5%. Produced from tumours;
roots spindly, <1 cm long.
9%. Produced from tumours

Ephedra Species

181

Fig. 12A-D. Production of tumours and/or roots on Ephedra following inoculation with
Agrobacterium rhizogenes. A One-year-old E. fragilis seedlings 35 days after injection with A.
rhizogenes strain Ar2629; X indicates tumours and roots at wound sites. B One-year-old E. fragilis
seedlings 35 days after injection with heat-killed A. rhizogenes strain Ar2629; arrows indicate
wound sites. C Typical dark orange tumour on 3-year-old E. fragilis plant following inoculation with
A. rhizogenes strain Ar1000. D Roots developing on the surface of internodal explants of E. fragilis
90 days after inoculation with A. rhizogenes strain Ar2629 on growth regulator-free medium

ration in quality or growth rate. Roots developed periodically from cultured


tumour tissue, but did not exceed 3 cm in length. Growth of these roots was
rapid initially but ceased after 3-4 days. However, in contrast to the behaviour
of tumorous tissue, sustained growth of excised roots was observed neither on

182

N.A. O'Dowd et al.

semisolid nor in liquid media. Two lines of E. fragilis tumorous tissue cultured
in the absence of growth regulators were regularly analysed for E, NE, PE and
NPE over a 2-year period. The faster-growing, friable line contained no alkaloid, but the slow-growing, compact line contained 0.01% E. Similarly,
Ar2629-induced E. minima tumorous tissues contained traces of PE (unpubl.).
Tumours formed on plants in the greenhouse could not be initiated into
culture, but they were analysed for alkaloid and compared to the stem from
which they derived. In general, tumours produced in vivo on the stems of
E. fragilis and E. minima plants contained levels of I-ephedrine similar to
those found in nontransformed stem tissue, but differed in that while
d-pseudoephedrine was detected in stem extracts, it could not be detected
in tumour extracts.
Studies involving the infection of Ephedra with Agrobacterium look promising. Tumour-derived tissues thus produced may provide a model system in
which alkaloid production could be investigated.
3.3 Organogenesis

Only two species of Ephedra have been successfully regenerated de novo in


vitro. Shoot regeneration was reported from diploid stem-derived callus of E.
gerardiana (Ramawat and Arya 1976; O'Dowd and Richardson 1993b) and E.
foliata (Arya and Ramawat 1988). E. gerardiana callus cultured on 0.05 pM
NAA and 0.03 pM kin became green, forming small, nodular structures after 8
months (O'Dowd and Richardson 1993b). On growth regulator-free medium,
about 50% of these structures developed into shoot meristerms. A small
number (2-3%) developed into normal stems (Fig. 13). Rhizogenesis also
occurred, but there was no evidence of a direct connection between the roots
and stem bases. Ramawat and Arya (1976) reported similar shoot differentiation from callus initially grown on 54 pM NAA and 0.4 pM kin and switched to
auxin-free medium supplemented optimally with 4pM kin or BAP. Shoot
production was observed from E. foliata calli grown on 0.5-2.3 pM IBA or 0.55.4 pM NAA (Arya and Ramawat 1988).
Regeneration of shoots has been reported from haploid female
gametophyte-derived explants of E. foliata (Konar and Singh 1979; Singh et al.
1981; Bhatnagar and Singh 1984). Shoots and roots grew out from meristemoids following optimisation of growth regulators in the medium. Optimal
shoot bud production in which 75% of cultures produced an average of 5.8
buds per explant was achieved on 2ppm kin with 0.5-2 ppm 2,4-D (Bhatnagar
and Singh 1984). It appears that, in general, high cytokinin levels supplemented with auxin are required for shoot morphogenesis, with kin and BAP
acting as the most effective cytokinins.
Kinetin and zeatin did not stimulate adventitious bud production.
E. saxatilis stem internodes produced shoot meristems on 10 pM BAP
and 5 pM 2,4-D but organogenesis was not observed for E. andina, E.
distachya, E. gerardiana, E. gerardiana var. sikkimensis or E. minima on this
medium.

Ephedra Species

183

Fig. 13. A Formation of adventitious shoot buds on E. gerardiana callus after culturing for 8
months on 0.05)1M N AA + 0.03)1 M kin. B The same callus after 2 months on growth
regulator-free medium showin g full development and branching of shoots

Another method of inducing adventitious shoot meristems in Ephedra was


demonstrated by germinating seeds on MS medium supplemented with 1020.uM BAP (O'Dowd and Richardson 1993b). Up to 20 20 shoot meristems
were obtained per seed on 20.uM BAP (Fig. 14). In order to allow shoots to
grow out, it is necessary to transfer them to a medium with low or zero growth
regulators, confirming earlier observations (Konar and Singh 1979; Singh et al.
1981).

N.A. O'Dowd et al.

184

Fig. 14. Adventitious shoot


buds developing from an
E. fragilis seed after 6
weeks' germination on
medium containing 20,LiM

BAP

3.4 Micropropagation
Ephedra is traditionally propagated by seed or vegetatively by the division of
older plants (Krishnamurthy et al. 1965; Morton 1977). Rooting of conventional cuttings has a low success rate. For example, Sanaenosuke and Kurihara
(1967) reported 20 and 3% rooting of cuttings from 4-year-old plants of E.
altissima and E. distachya, respectively. An efficient micropropagation system
offers the opportunity to rapidly increase elite strains (e.g. high alkaloidyielding) and to speed up the production of select Ephedra plants from conventional hybridisation or mutagenesis. Prior to our own studies (O'Dowd and
Richardson 1993a), only two Ephedra species, E. foliata and E. gerardiana,
had been micropropagated in vitro and rooting had been problematic
(Sankhla et al. 1967b; Ramawat and Arya 1976; Konar and Singh 1979;
Bhatnagar and Singh 1984; Arya and Ramawat 1988).
O'Dowd and Richardson (1993a) studied ten Ephedra species in an attempt to develop a general micropropagation protocol for the genus. There
were differences in performance between species but in several cases some
species were represented by only one parent individual. In preliminary studies,
nodal stem explants of E. fragilis were cultured on MS medium supplemented
with 3% sucrose, 0.05,uM IBA and 0-5,uM BAP, kin or zea. Typically, with
increasing cytokinin levels the number of shoots per explant increased while
shoot length decreased (Fig. 15). An optimal cytokinin concentration of
0.05,uM kin was chosen, giving a micro propagation coefficient of 5 per 30-40
days while maintaining shoot quality. Nine other Ephedra species were similarly grown on this medium and the results are summarised in Table 10.
Alkaloids were not found in shoot cultures from any of the alkaloid-yielding
Ephedra species (O'Dowd 1991).

(n = 76)
E. scoparia
(n = 79)
E. viridis
(n = 35)

E. saxatilis

(n = 98)

E. minima

E. andina
(n = 102)
E. equisitina
(n = 153)
E. fragilis var
camplyopoda
(n. ::':: 54)
E. gerardiana
(n = 69)
E. intermedia
(n = 49)
E. major
(n = 54)
E. major ssp.
procera
(n = 51)

Species

33

24

29

13

11

27

21

21

47

100

18.0 ::':: 4.7

40

60

42.5 ::':: 14.2

72

34.0 ::':: 11.0

14.8 ::':: 7.3

28

29

6.2::':: 5.3

24.9 ::':: 12.0

27.9::':: 8.6

12.4 ::':: 5.9

68

58

87

60

43

33

17.5::':: 6.5

42
50

22.0 ::':: 7.5

Explants with
3-4 shoots (%)

56

Explants with
2 shoots (%)

Averafte
shoot ength

32

13

13

40

57

48

20

17

13

29

44

Explants with
1 shoot (%)

As a percentage of surviving explants

58

12

Non-growin,
explants (%

58

14

Dead
explants (%)

Shoots hcalthy, medium


green
Shoots generally pale with
callus forming at the nodes
Short stems with swollen
nodes
Stem tips red, with high
number of nodes (~4).
Many vitrified and forming
callus
Shoots healthy, pale green
and slender (0.8 mm in
diameter)
Shoots healthy, mediumgreen (1.2mm in diameter)
Shoots pale, most were
vitrified
All explants brown and dead

Shoots medium green. some


vitrification of shoot bases
Explant nodes brown. Shoots
pale green
~ 75 % of shoots vitrified,
some shoot tips red-pink

Additional notes

Table 10. Shoot production from stem nodal explants of ten species of Ephedra after 40 days on MS medium supplemented with 0.05}1M lEA and 0.05}1M
kin. n = No. of explants cultured

00
U1

f-'

V>

(D'

(1)

'"'"'

C/l

.,.'"

~
~

186
30

E
-5
.s

N.A. O'Dowd et al.

---

20

Fig. 15A,B. The effects of


increasing cytokinin concentration on A shoot length and B
the number of shoots produced per E. tragilis nodal
explant. Standard errors given,
n = 20. (After O'Dowd and
Richardson 1993b)

01)

s::

,j,l

00

..c:

10

CI'J

1.0

2.0

3.0

4.0

5.0

cytokinin concentration (I-lM)

---

30

---0--

0.. 20

BAP
KIN

~
<l)

...

<l)

0..

~0

..c:
</J

10

0
0

1.0

2.0

3.0

4.0

5.0

cytokinin concentration (I-lM)

Healthy shoot cultures of E. andina, E. equisitina, E. tragilis, E.


gerardiana, E. minima, E. minima hybrid (a possible interspecific cross between E. minima and E. gerardiana - O'Dowd 1991) and E. saxatilis were
established. Rooting was poor on agar-solidified medium compared to medium-saturated Sorbarods (O'Dowd and Richardson 1993a). Basal callus and
hyperhydricity were often associated with NAA (0.1-5 pM) or high levels of
IBA (>5 pM) in the rooting medium. Shoots from high cytokinin-containing
media tended to root poorly. Best rooting was achieved on 5 pM IAA. In fact,
it was possible to achieve a single-stage protocol for the production of plantlets
of E. equisitina, E. minima hybrid, E. gerardiana and E. saxatilis in Sorbarod
chambers by hedging every 30-40 days on this medium.
Healthy roots were a prerequisite for weaning, which was carried out in
Sorbarod chambers containing a mix of John Innes no.1 compost:perlite (1: 1)
for good aeration. Roots failed to produce root hairs in pure soil and eventually rotted. Captan treatment (1.9 gl-l) was necessary to prevent fungal attack.
Benlate caused chlorosis and death of plantlets. Rates of up to 100% acclimatization were possible by this method.
In summary, it is now possible to micropropagate four species of Ephedra
using a single-stage protocol. This could provide a useful method for quickly

Ephedra Species

187

producing large numbers of clonal plants for the production of superior


genotypes.
3.5 In Vitro Induction of Haploids

The production of haploid tissues and plants from Ephedra has been reported.
For example, Konar (1963) cultured haploid tissues in vitro from E. foliata
pollen. The production of haploid callus from the E. foliata female
gametophyte, and the subsequent regeneration of haploid plantlets was reported by Singh et al. (1981) and Bhatnagar and Singh (1984). They found that
the female gametophyte was more easily manipulated in vitro than the male
gametophyte. Ovules were harvested when the pollination drop was present,
as this indicates that the archegonia are mature. Singh et al. (1981) described
optimal conditions for callus and root production from mature archegonia
stage ovules on 9 flM 2,4-D or 22 flM N AA. They also demonstrated the ability
of these tissues to regenerate shoots. Shoot bud formation was stimulated by
the addition of BAP (36 flM) or a combination of 2,4-D (9 flM) and kin (528flM) to the medium. Subsequent transfer to growth regulator-free medium
was essential to allow further bud development. Observation of chromosome
numbers showed these plants to be haploid (n = 7).
These techniques for the production of haploid Ephedra plants could be
used to produce mutants which, for example, overproduce alkaloid, or have a
different ratio of alkaloids, or in which the biosynthetic pathway has been
disrupted. Such mutants might provide useful information on the secondary
metabolite biosynthetic pathway of the genus.

4 Summary and Conclusion


Our interest was to establish whether an enzyme(s) exists which interconverts
ephedrine and pseudoephedrine, hence the choice of Ephedra for our studies.
The infection of Ephedra with Agrobacterium rhizogenes led to the production
of tumorous tissues capable of low-level, but stable, alkaloid production.
Further work in this direction could prove beneficial in establishing a model
experimental system.
Kutchan (1995) states that many of the problems with elucidating plant
secondary biosynthetic pathways have often stemmed from the complexity of
the molecules being studied. With Ephedra the problems are almost the reverse. Due to the relative simplicity of the ephedrine alkaloid molecules and
their apparent similarity to many compounds produced by both parent plant
and in vitro cultures, it was difficult to monitor their production. In our
experience, HPLC/mass spectroscopy could not distinguish several compounds isolated from collected HPLC fractions or TLC spots. A more specific
method for the identification of these compounds is required. We raised

188

N.A. O'Dowd et al.

antibodies to E and PE for identifying the presence of alkaloids in plant,


culture and cell-free extracts and for locating them in plant sections and cells.
An ELISA assay for the determination of ephedrine was developed (unpubl.),
but further work would be required to clean up the antiserum to ensure its
monospecificity towards ephedrine. Using radiolabelled Ephedra alkaloids
may reveal whether interconversion occurs between the alkaloids and could
help trace the fate of any intermediates. It may prove unnecessary to develop
an in vitro system by applying such radiolabelled compounds through the base
of cut stems or by painting them onto the stem surface. This approach, using
intermediates from the early steps of the pathway, has yielded most of the
current information on E/PE biosynthesis.
The problems with elucidating the ephedrine/pseudoephedrine biosynthetic pathway are twofold. Firstly, there is the need for a system in
which the alkaloids are being actively biosynthesised and to which precursors
and cofactors can be added and in which the flux of compounds can be traced.
Secondly, there is a need to develop systems for separating, isolating and
identifying the compounds involved. An in vitro system capable of producing
ephedrine and pseudoephedrine at commercial levels from Ephedra cultures is
not possible at present. What is sought is a system capable of detectable
alkaloid production, even at a low level, as long as it is reliable. This would
offer a model suitable for enzyme studies to elucidate secondary biosynthetic
pathways. Theoretically, this is easier in vitro, as cultured tissues are more
homogeneous, contain fewer phenolics, and precursor or elicitor feeding and
environmental conditions are more easily controlled. If it proves possible
to identify enzymes catalysing the reactions in the ephedrine and pseudoephedrine biosynthetic pathway, it may become possible to isolate DNA encoding these proteins. The problem has been to develop cultures which
reliably produce ephedrine and pseudophedrine. Reevaluation of culture conditions and media is one possibility.
The study of alkaloid biosynthesis has recently started to fulfil some of the
early promises. Biosynthetic pathways are being characterised, e.g. tropane
alkaloids from several Solonaceae species (Yamada and Hashimoto 1988),
benzophenanthridine alkaloid synthesis in Eschscholtzia californica (Kutchan
and Zenk, 1993). The cDNA for enzymes from plant secondary biosynthetic
pathways are being expressed in other organisms, e.g. strictosidine synthase
from Rauwolfia serpentina, has been expressed in Escherichia coli (Kutch an
1989); the berberine bridge enzyme from Eschscholtzia californica has
been expressed in insect cell culture (Kutchan et al. 1994). If it proves
possible to identify enzymes catalysing the reactions in the I-ephedrine and
d-pseudoephedrine biosynthetic pathway, it may subsequently lead to the
isolation of cDNA encoding these proteins. It has been envisaged that
such DNA could be inserted into the bacterial genome and be expressed.
With Ephedra, it may be sufficient to express only the final step or few steps of
the alkaloid biosynthetic pathway in a fermentation system. Cheap, bulk precursors could be fed to the system or cheap ephedrine to a system that converts
it to the more valuable pseudoephedrine (if such a step is enzymatically
catalysed).

Ephedra Species

189

Such approaches are commercially valid where the product is of high


value and the market demand hard to satisfy from natural sources, especially
where chemical synthesis is not a viable option. Novel compounds from in
vitro systems could be similarly exploited. Each case requires evaluation upon
its own merits. In the case of Ephedra we are dealing with pharmaceutical
compounds which command a low price but are of value on account of the size
of the market. With a growing world population and a growing per capita
demand for medicines, the ephedrine/pseudoephedrine market will probably
continue to expand, especially in its role as an anti asthmatic. Producing
Ephedra alkaloids by a fermentation system may prove necessary if demand
outstrips the rate of production by current means.
Studies to improve natural methods of production should parallel
biotechnological research. In this and other studies, protocols have been
established for improved identification of alkaloid profiles in plants. High
alkaloid-yielding or high pseudoephedrine-producing individuals can be identified or, perhaps, engineered by molecular techniques, e.g. by preventing
catabolism or blocking competitive pathways. Micropropagation methods
could then be used to produce large numbers of clones to supplement the
alkaloid yield of the natural population. This approach could improve the
economics of the natural production of the ephedrine alkaloids.
Acknowledgements. The authors thank Prof. David Richardson, Dr. Sheila McNally, Devon
Thompson, Drs. Gordon Provan, Una Hearty, John Rice. Marcel Jaspars and Peter Owen for
their input to the "Ephedra project". Dr. Conor O'Brien and Avondale Chemical Co. and the
Industrial Development Authority (Ireland) for their financial support and advice. Thanks also to
the School of Botany, Dept. of Genetics. and Botanic Gardens of Trinity College Dublin and the
School of Botany, University College Dublin. Also the Botanic Gardens of Marburg and
Wurzburg Universities. Kew. and Dr. Mark Seaward, Leeds Univ. are acknowledged for the
provision of plant material.

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XI Euglena gracilis Z: Biotransformation of


Terpenoids and Related Compounds
Y. NOMA! and Y. ASAKAWA 2

1 Introduction
Euglena was first discovered by Antony van Leeuwenhoek in 1675 and is
called Midori mushi in Japanese. The name Euglena means beautiful (Eu) eye
(glena). Euglena is classified in both the animal and the plant kingdoms. In the
animal kingdom Euglena belongs to the Protozoa, whereas in the plant kingdom Euglena belongs to Euglenophyta (Inoki 1981; Yamada et al. 1983).
There are more than 60 species belonging to the Euglena genus, of which
about 10 are well known (Johnson 1968; Mizuno 1976; Inoki 1981). Euglena is
considered as having stable heredity because it has no sex and no reduction
division. Cell sizes of Euglena vary from ca. lO.um (E. min uta ) to ca. 500.um
(E. oxyuris). The cell shape is very variable due to a metabolism called
euglenoid movement; being spindle-shaped and spherical, the cells may
change their shape under changing conditions. Euglena gracilis, especially
strain Z and var. bacillaris, are widely used for investigations in physiology and
biochemistry. Euglena gracilis is a unicellular microorganism (ca. 50.um in
length x ca. lO.um in width), which has chloroplasts, mitocondria, and two
flagella of different lengths, an eye spot containing carotenoid and flavin, and
other organelles such as Golgi bodies. Euglena can move by swimming, or
contracting to changing shapes and crawling and sliding; most of the species
have a forward rotary movement by swimming spirally. Euglena has the
paramylon, f3-1,3-glucan, as one of the storage substances. At first, only few
biologists were interested in Euglena as a rare microorganism. However, since
the utilization of Euglena as a test microorganism both for the measurement of
vitamin BI2 biologically in 1949 and the establishment of the Calvin-Benson
cycle in photosynthesis in the 1950s, it has become as a very important microorganism for the investigation of photosynthesis and chloroplast. Now, many
biologists, physiologists, and biochemists are interested in Euglena regarding
its utilization in the investigation of physiological and biochemical phenomena
as a small animal and a small plant. Furthermore, Euglena has characteristic
features in the TCA cycle, wax ester fermentation, and de novo fatty acid

I Faculty of Domestic Sciences, Tokushima Bunri University, Yamashiro-cho, Tokushima 770,


Japan
2 Faculty of Pharmaceutical Sciences, Tokushima Bunri University, Yamashiro-cho, Tokushima
770,Japan

Biotechnology in Agriculture and Forestry, Vol. 41


Medicinal and Aromatic Plants X (ed. by y'P.S. Bajaj)
Springer-Verlag Berlin Heidelberg 1998

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195

synthesis by adverse j3-oxidation. In industry, Euglena is used not only for cells
as nutrients (Hosoya and Kitaoka 1977; Kitaoka and Hoyoya 1977) and soluble paramyron for its anticancer effect (Yokota, unpubl), but also for commercial manufacture of torehalose from glucose by trehalose phosphorylase
(Murao et al. 1985), arachidonic acid (Sudate and Goto 1986), wax ester (Tani
et al. 1987), and vitamin E production (Tani and Tsumura 1989). The biochemistry, physiology, subcellular biochemistry, and molecular biology of Euglena
has been collected in The Biology of Euglena (Buetow 1968, 1982, 1989).

2 Cultivation
Euglena is widely distributed worldwide and lives in nonflowing water such as
ponds and swamps. It moves between the surface and the bottom of the ponds,
in search of the most comfortable living conditions. Euglena is also capable of
living in natural environments with a wide range of light, temperature, pH,
soluble Oz, and soluble COz; it is also found in animal- and plant-derived
sewage, wastewater from food processing factories, and strong acidic
wastewater from mines. Several media such as Cramer-Myers, Hutner, and
Koren-Hutner, are used for the cultivation of Euglena (Kitaoka 1989). In our
studies, Hutner medium was used (Schiff et al. 1971). Euglena gracilis Z (wildtype) was cultivated under stationary conditions at 2S C for 7-10 days under
both photoheterotrophical and photoautotrophical conditions (light illumination ca. 2000-3000Ix) in SOOml of Hutner medium in an Erlenmeyer flask
containing KH zP0 4 (O.4g), (NH 4 )zHP04 (0.2g), MgS0 47HzO (O.Sg), CaC0 3
(0.2g), DL-malate (2g), Na-glutamate (S g), EDT A 2Na (SOmg), ZnS0 47H20
(22mg), MnS0 42H20 (S.8mg). FeS0 4 (NH4)2S046HzO (S.7mg), Na 2Mo0 4
H 20 (l.Smg), CuS0 4SH20 (1.6mg), CoS0 47H zO (l.Smg), H3B04 (11.4mg),
vitamin B) (2.5mg) and vitamin Bl2 (O.02mg) in 11 distilled water (pH 3.3,
adjusted with HCI). Euglena gracilis SM-ZK, a streptomycin-bleached mutant
derived from strain Z. was also cultivated under stationary conditions and
heterotrophically in the dark. Euglena gracilis Z grows even under
photoautotrophical, photoheterotrophical, and heterotrophical conditions.

3 Biotransformation of Terpenoids and Related Compounds


We are continuing study on the biotransformation of plant secondary
metabolites and synthetic compounds in microorganisms (Noma and Asakawa
1994, 1995) and chose Euglena gracilis Z (kindly supplied by Professor emeritus S. Kitaoka, University of Osaka Prefecture), to study the biotransformation of monoterpenoids and related compounds (Noma 1987;
Noma et al. 1989, 1990, 1991a,b, 1992a,b, 1993, 1994a,b, 1995; Noma and
Asakawa 1992). On obtaining full growth of Euglena, we carried out
biotransformation of terpenoids and related compounds at ca. SO-200 mg!l

y. Noma and Y. Asakawa

196

(average 100mg/l) for 1-30 days under the same conditions as described
above. For time course changes, aliquots (3 ml) of the cultured broth were
taken and put on the an Extrelut-column and extracted with 6ml of Et 20.
Each ether extract was analyzed by GC-MS. For aquisition of metabolites,
large-scale culture was carried out repeatedly, and the broth was extracted
with Et 20 after the removal of Euglena by centrifugation. The total recovery
ratios of metabolic products were more than 90%. The metabolites were
separated and purified by a combination of CC on silica gel, preparative GC,
Sephadex LH-20, and/or prep. HPLC. The products were identified by comparison with GC (capillary column, 30m X 0.25mm i.d., CDX-B, 13cyclodextrin/O V -1701) retention times, mass spectrometry, IR, 1H -( 400 MHz),
and 13C-NMR (100MHz) spectra with those of authentic specimens.
Hydrocarbons

Terpene aldehydes

Terpene ketones

Terpene epoxides

100

.~

'">

.~

50

o;,~-Unsaturated

ketones

abc d e f g h

k I mn

Saturated
ket('nes

p q r s t u v wx Y zo;

Compounds

Fig. I. Relationship between structure of terpenoids and survival of Euglena gracilis Z after 1 day.
(Noma et al. 1991b)
Hydrocarbons: a d-limonene(136'); b p-menthane; c a-pinene; d j3-pinene; e longifolene;
f caryophyllene
Terpene aldehydes: g d-citronellal(38); h l-citronellal(39); i dl-citronellal(38 and 39); j citral
[geranial(30) and neral(31)]; k l-perillaldehyde(7a); I l-phellandral(18); m
trans- and cis-1,2-dihydroperillaldehydes(14 and 15); n cumin aldehyde(27);
o myrtenal(l)
Other aldehydes: p cinnamic aldehyde(96)
Terpene ketones:
a,j3-Unsaturated ketones: q l-carvone(123); r d-carvone(123'); s dl-piperitone;
t isopiperitenone; u n-piperitenone; v verbenone
Saturated ketones: w dl-isomenthone; x I-menthone; y mixture of d-dihydrocarvone(124)
and isodihydrocarvone(12S); z mixture of l-dihydrocarvone(124') and
isodihydrocarvone(12S')
Terpene epoxides: a l,4-cineole; f31,8-cineole; ytrans- and cis-shisool-8,9-epoxides

Euglena gracilis Z

197

The relationship between 29 terpenoids and the survival of Euglena cultured photo heterotrophically for 1 day was investigated by measuring absorption at 640nm as turbidity; the result is shown in Fig. 1 (Noma et al. 1991b).
Most terpene aldehydes, aromatic aldehyde, and terpene ketones showed
strong inhibition for the survival of Euglena et ca. 200ppm. Table 1 and Fig. 2
Table 1. Summary of biotransformation of mono terpene aldehydes by Euglena. (Noma et al.
1991b)
Substrates

Products [" T.r. %]

Myrtenal (1)

Myrtenol (2. M')


Myrtenoic acid (3, me)
(IS)-Myrtanol (4a, M)
(lS)-Myrtanoic acid (6a, m)
(1 R)-Myrtanol (5b. M)
(1 R)-Myrtanoic acid (6b, m)
I-Perillyl alcohol (8a, M)
trans-Shisool (9, M)
cis-Shisool (10, m)
trans-Shisoic acid (16, M)
I-Perillic acid (13a, m)
8-Hydroxy-trans-shisool (11, M)
H-Hydroxy-cis-shisool (12, m)
dl-Perillyl alcohol (8a and b, M)
trans-Shisool (9, M)
cis-Shisool (10, m)
8-Hydroxy-trans-shisool (11, M)
8-Hydroxy-cis-shisool (12, m)
I-Phellandrol (19, M)
trans-Tetrahydroperillyl alcohol (21, M)
cis-Tetrahydroperillyl alcohol (22, m)
I-Phellandric acid (20, m)
trans-Tetrahydroperillyl alcohol (21, M)
trans-Tetrahydroperillic acid (25, m)
cis-Tetrahydroperillyl alcohol (22, M)
cis-Tctrahydroperillic acid (26, m)
trans-Shisool (9, M)
cis-Shisool (10, M)
trans-Shisoic acid (16, M)
cis-Shisoic acid (17, m)
Cumin alcohol (28, M)
Cuminic acid (29, m)
Geraniol (32, M)
Nerol (33, M)
d- or I-Citronellol (34 or 35, M)
d-Citronellol (34, M)
d-Citronellic acid (40, m)
p-Menthane-3,8-trans-diol (42, M)
p-Menthane-3,8-cis-diol (43, M)
I-Citronellol (35, M)
I-Citronellic acid (41, m)
p-Menthane-3,8-trans-diol (44, M)
p-Menthane-3,8-cis-diol (45, M)
dl-Citronellol (34 and 35, M)
dl-Citronellic acid (40 and 41, m)
p-Menthane-3,8-trans-diol (42 and 44, M)
p-Menthane-3,8-cis-diol (43 and 45, M)

(lS)-Myrtanal (5a)
(lR)-Myrtanal (5b)
I-Perillaldehyde (7a)

dl-Perillaldehyde (7a and b)

I-Phellandral (18)

trans- and cis- Tetrahydroperillaldehydes (23 and 24)

trans- and cis-I,2-Dihydroperillaldehydes (14 and


15 = 83: 17)

Cumin aldehyde (27)


Citral[Geranial (30) and
Neral (31) = 56: 44]
d-Citronellal (38)

I-Citronellal (39)

dl-Citronellal (38 and 39)

, T.r. transformation ratio.


b D.C. lethal concentration of substrate for Euglena.
, M Major product.
" m minor product.

D.C.
E.g.
120

Cui or mgl1 00 ml)


E.w.
100

E.a.
100

50

50

40

50

50

60

70

50

30

100

100

110

50

50

40

70

80

100

180

40

150

180

190

180

180

130

160

GHO

G-- G

~'
GOOH

GHO

~- ~
3

~'

--+'

4a

5a

6a

* liJ GJ -- GJ
gH 20H

GOOH

gOOH
,

~HO

4b

5b

8'

6 GHO

14

GOOH

2~
13b

2 2'
GHO

7b

8b

)(OH
11

6 - 6 6'
-0
A
A
GHO

yH 20H

13a

7a

8a

GHO

0
A

15

16

GOOH

cS' 0'
A

"-

6b

OH

12

10

---

0'
A

"-

0'
A

17

Fig. 2. Biotransformation of monoterpene aldehydes by Euglena gracilis Z. Compounds: 1


myrtenal; 2 myrtenol; 3 myrtenoic acid; 4a lR-myrtanol; 4b lS-myrtanol; Sa lR-myrtanal; 5b lSmyrtanal; 6a lR-myrtanoic acid; 6b lS-myrtanoic acid; 7a I-perillaldehyde; 7b d-perillaldehyde; 8a
I-perillyl alcohol; 8b d-perillyl alcohol; 9 trans-shisool; 10 cis-shisool; 11 8-hydroxy-trans-shisool;
12 8-hydroxy-cis-shisool; 13a I-perillic acid; 13b d-perillic acid; 14 trans-l ,2-dihydroperillaldehyde;
15 cis-l,2-dihydroperillaldehyde; 16 trans-shisoic acid; 17 cis-shisoic acid; 18 I-phellandral; 19 1phellandrol; 20 I-phellandric acid; 21 trans-tetrahydroperillyl alcohol; 22 cis-tetrahydroperillyl
alcohol; 23 trans-tetrahydroperillaldehyde; 24 cis-tetrahydroperillaldehyde; 25 transtetrahydroperillic acid; 26 cis-tetrahydroperillic acid; 27 cumin aldehyde; 28 cumin alcohol; 29
cuminic acid; 30 geranial; 31 neral; 32 geraniol; 33 nerol; 34 d-citronellol; 35 I-citronellol; 36
geranic acid; 37 neric acid; 38 d-citronellal; 39 I-citronellal; 40 d-citronellic acid; 41 I-citronellic
acid; 42 d-p-menthane-trans-3, 8-diol; 43 d-p-menthane-cis-3,8-diol; 44 l-p-menthane-trans-3,8diol; 45 l-p-menthane-cis-3,8-diol. (Noma et al. 1991b)

199

Euglena gracilis Z

(s~~
...............
20

2)

21

23

~
25

18

2
COOH

--

29

28

27

~w
I

31

~ooc
37

Fig. 2. Continued

33

35

CHO

--z

OH
OH

39

45

::

0~

COOH

41

n"'OH
XOH

44

200

y. Noma and Y. Asakawa

show biotransformations of terpene aldehydes and a lethal concentration


of substrate for Euglena cultured photoheterotrophically (Eg.), heterotrophically (E w.; strain SM -ZK), and photoautotrophically (Ea.), when it was
added continuously to the medium every day (Noma et al. 1991b). When
myrtenal (1) was added to the cultured broth of Euglena, it was reduced to
give myrtenol (2) as the major product and myrtenoic acid (3) as the minor
product. However, compound 2 was not further hydrogenated to myrtanol (4a
and 4b) even at a concentration less than ca. 50mg/l. (lS)- and (lR)-Myrtanal
(5a and 5b) were also transformed to 4a and 4b as the major product and (lS)and (lR)-myrtanoic acid (6a and 6b) as the minor product, respectively. Compound 7a was also easily transformed to give I-perillyl alcohol (Sa) and transshisool (9), which is well known as a fragrance, as the major products, and
I-perillic acid (13a) as the minor product (Table 1, Fig. 2). Compound Sa was
also transformed to 9 as the major product with cis-shisool (10) and 8-hydroxycis-shisool (12) as the minor products. Further, compounds 9 and 10 were
hydroxylated to 8-hydroxy-trans-shisool (11) and 12, respectively. trans- and
cis-1,2-Dihydroperillaldehydes (14 and 15) were also transformed to 9 and 10
as the major products and trans- and cis-shisoic acids (16 and 17) as the minor
products, respectively. Compound 16 was also formed from 9. In the
biotransformation of dl-perillaldehyde (7a and 7b), the same results were
obtained as described in the case of 7a. I-Phellandral (IS) was metabolized
mainly via I-phellandrol (19) to trans-tetrahydroperillyl alcohol (21). transand cis-Tetrahydroperillaldehydes (23 and 24) were also transformed to 21
and cis-tetrahydroperillyl alcohol (22) as the major products and trans- and cistetrahydroperillic acids (25 and 26) as the minor products, respectively.
Cuminaldehyde (27) with an aromatic ring was also transformed to cumin
alcohol (2S) as the major product and cuminic acid (29) as the minor product.
Citral [a mixture of geranial (30) and neral (31), 56:44 peak area in GC] was
easily transformed to geraniol (32) and nero I (33), respectively, of which 32
was further hydrogenated to citronellol (34 or 35) (Table 1, Fig. 2). Geranic
acid (36) and neric acid (37) as the minor products were also formed from 30
and 31, respectively. On the other hand, when either 32 or 33 was used as a
substrate, both compounds were isomerized to each other and, then, 32 was
transformed to citronellol (34 or 35). These results showed that Euglena could
distinguish between the stereoisomers, 32 and 33, and hydrogenated selectively 32 to citronellol (34 or 35). d-, 1- and dl-Citronellal (3S, 39, and equal
mixture of 3S and 39) were also transformed to the corresponding d-, 1-, and dlcitronellol (34, 35, and equal mixture of 34 and 35) and p-menthane-trans- and
cis-3,8-diols (42-45) as the major products, which are known as mosquito
repellents and plant growth regulators (Nishimura et al. 1982, 1986), and d-, I,
and dl-citronellic acids (40, 41, and equal mixture of 40 and 41) as the minor
products, respectively. Terpene aldehydes tested here were reduced mainly to
the primary terpene alcohols at a concentration of ca. 100ppm with the minor
corresponding terpenic acids. a,j3-Unsaturated terpene aldehydes such as 7a,
IS, dl-perillaldehyde (equal mixture of 7a and 7b), 30, and 31 were firstly
reduced to the corresponding primary a,j3-unsaturated terpene alcohols,
which were further hydrogenated at the a,j3-unsaturated C = C double bond

201

Euglena gracilis Z

to give the saturated primary terpene alcohols. In the biotransformation of


a,/3-unsaturated terpene alcohols such as 8a, 19, dl-perillyl alcohol (8a and 8b),
selective hydrogenation occurred from either the re-face to C-l for 8a and 19
or the si-face for d-perillyl alcohol, and 9 and 21 were predominant, respectively (Fig. 3).
The biotransformation of unsubstituted aromatic aldehyde benzaldehyde
(46), the monosubstituted aromatic aldehydes 0-( =salicyl aldehyde), m-, and
p-hydroxybenzaldehyde (47-49), 0-, m-, and p-anisaldehyde (50-52), 0-, m-,
and p-chlorobenzaldehyde (53-55), 2-, 3-, and 4-cyanobenzaldehyde (56-58),
0-, m-, and p-nitrobenzaldehyde (59-61), 0-, m-, and p-tolualdehyde (62-64),
0- and terephthalaldehydic acid (65 and 66), 0-, iso-, and terephthalaldehyde
(67-69), the disubstituted aromatic aldehydes o-vanillin (70), 3nitrosalicylaldehyde (71), vanillin (72), veratraldehyde (73), isovanillin
(74), ethylvanillin (75), 2-hydroxy-5-methylbenzaldehyde (76), 2,4dimethylbenzaldehyde (77), 2,4-, 2,5-, and 3,4-dihydroxybenzaldehyde (7880), 2,3-, 2,4-, 2,5-, and 3,5-dimethoxybenzaldehyde (81-84), 2,3-, 2,4-, 2,6-,
3,4- and 3,5-dichlorobenzaldehyde (85-89), the trisubstituted aromatic aldehydes 2,3,4-, and 3,4,5-trimethoxybenzaldehyde (90 and 91), and other related
aldehydes, p-acetylamidebenzaldehyde (92), phenylacetaldehyde (93), 2- and
3-phenylpropionaldehyde (94 and 95), cinnamic aldehyde (96), a-methyl cinnamic aldehyde (97), nicotin aldehyde (98), aliphatic aldehydes n-heptanal
(99), pelargonaldehyde (100), trans-2-hexenal (101), trans-2-heptenal (102),

Substrates

Major products

I-Perillyl alcohol(8a)
dl-Perillyl alcohol(8a and b)
I-Phellandrol(19)

trans-Shisool(9)
tralls-Shisool(9)
trans-Tetrahydroperillyl alcohol(21)

lsopropenyl
Isopropenyl
Isopropyl

Fig. 3. Selective hydrogenation of I-perillyl alcohol(8a). dl-perillyl alcohol(8a and 8b), and 1phellandrol(19) by Euglena gracilis Z. (Noma et al. 1991b)

202

y. Noma and Y. Asakawa

cis-4-heptenal (103), trans-2-decenal (104), 2,4-hexadienal (105), trans, trans2,4-heptadienal (106) and trans, trans-2,4-nonadienal (107) is summarized in
Table 2 together with the substrate concentration for the survival of the
phytofiagellate (Noma et al. 1991a). The biotransformation yield of aromatic
aldehydes and related aldehydes was ca. 100% after 1 or 2 days. Most of the
aromatic, aliphatic, and related aldehydes were transformed to the corresponding primary alcohols as major products. However, some of them gave
the corresponding acids as major or minor products. m-Chlorobenzaldehyde
(54) was transformed to m-chlorobenzyl alcohol (108) as the major product
and m-chlorobenzoic acid (109) as the minor product. However, the content of
108 decreased gradually with time and eventually 109 became predominant. pChlorobenzaldehyde (55) was strongly phytotoxic at a concentration of
100 ppm. However, it was slightly transformed to p-chlorobenzyl alcohol as the
major product and p-chlorobenzoic acid (110) as the minor product. After the
death of the Euglena cells, the predominant product became 110. The order of
preference for the formation of chlorobenzoic acids from 53-55 by Euglena
was 54 and 55. Compound 53 was not oxidized to the acid. 2Cyanobenzaldehyde (56) was transformed first to 2-cyanobenzyl alcohol,
which was further transformed via imine to phthalide (111) as the final product. On the other hand, o-phthalaldehydic acid (65) and o-phthalaldehyde (67)
were also transformed to 111. 0-, m-, and p-Tolualdehyde (62-64) gave the
corresponding alcohols as the major products together with the formation of
small amounts of toluic acid in the cases of 63 and 64. The reduction of iso- and
terephthalaldehyde (68 and 69) was carried out stepwise with the corresponding aldehydic alcohols as intermediates and iso- and terephthalalcohol as the
final products. When ca. 100mg of compounds 70-76 were added into 500ml
of cultured broth day by day, final concentrations of 70, and 72-76 were 0.12,
0.26,0.40,0.44,0.12, and 0.13%, respectively, at which Euglena died; they were
completely transformed to the corresponding alcohols (112, 114-118) (Fig. 4).
On the other hand, although compound 71 was very phytotoxic, it was transformed to 113 at low concentration, i.e., below 100mg/l. The velocity of
biotransformation for 72 and 75 to 114 and 117, respectively, in the light
(ca. 3000 Ix) by Euglena cultured photoheterotrophically was faster than in
the dark (Fig. 5). Furthermore, compound 72 was easily biotransformed to
114 in the cell-free extract of Euglena. Compounds 93--97 were also transformed to the corresponding primary alcohols. In the cases of the unsaturated
alcohols such as cinnamyl alcohol (119) and 2-methylcinnamyl alcohol (121),
the C = C double bond was hydrogenated to give the corresponding saturated alcohol (120 and 122). 3-Phenylpropanol (120) was subsequently
oxidized to 3-phenylpropionic acid via 3-phenylpropionaldehyde (95).
p-Acetylamidebenzaldehyde (92) and nicotin aldehyde (98) as nitrogencontaining aromatic aldehydes were also reduced to the corresponding
alcohols. However, compound 98 was extremely phytotoxic at ca. 100ppm. In
the present method only one container is used and has a short time reaction;
the preparation is very simple. On the basis of the above results, we consider
that Euglena is a good bioreactor for the reduction of aldehydes to the corresponding primary alcohols.

203

Euglena gracilis Z

Table 2. Summary of biotransformation of benzaldehyde and related compounds by Euglena.


(Noma et al. 1991b)

bD.C. CuI or mg/100ml)


Substrates
Benzaldehyde (46)
o-Hydroxybenzaldehyde (47,
Salicylaldehyde)
m-Hydroxybenzaldehyde (4S)
p-Hydroxybenzaldehyde (49)
o-Anisaldehyde (50, o-MeO)
m-Anisaldehyde (51, m-MeO)
p-Anisaldehyde (52, p-MeO)
o-Chlorobenzaldehyde (53)
m-Chlorobenzaldehyde (54)
p-Chlorobenzaldehyde (55)
2-Cyanobenzaldehyde (56)
3-Cyanobenzaldehyde (57)
4-Cyanobenzaldehyde (5S)
o-Nitrobenzaldehyde (59)
m-Nitrobenzaldehyde (60)
p-Nitrobenzaldehyde (61)
o-Tolualdehyde (62)
m-Tolualdehyde (63)
p-Tolualdehyde (64)
o-Phthalaldehydic acid (65)
Terephthalaldchydic acid (66)
o-Phthalaldehyde (67)
Isophthalaldehyde (68)
Terephthalaldchyde (69)
o-Vanillin (70)
3-Nitrosalicylaldehyde (71)
Vanillin (72)
Veratraldehyde (73, 3,4dimethoxybenzaldehyde)
Isovanillin (74)
Ethylvanillin (75)
2-Hydroxy-5methoxybenzaldehyde (76)
2,4-Dimethylbenzaldehyde (77)
2,4-Dihydroxybenzaldehyde (7S,
f3- Resorcyl aldehyde)
2,5-Dihydroxybenzaldehyde (79,
Gentisinaldehyde)

Products (" T.r., %)


Benzyl alcohol (22%, M')
Benzoic acid (13'1'0, m d)
Salicyl alcohol (M)

'E.g.

fE.a. gE.w.

60

10 30

80

10 30

114
273
80
40
40
30

m-Hydroxybenzyl alcohol (M)


p-Hydroxybenzyl alcohol (M)
o-Anisalcohol (100% I day)
m-Anisalcohol (100% 1 day)
p-Anisalcohol (90% 1 day)
o-Chlorobenzyl alcohol
(100% 1 day)
m-Chlorobenzyl alcohol
(10S,49% I day)
m-Chlorobenzoic acid (109, M)
p-Chlorobenzyl alcohol (M)
p-Chlorobenzoic acid (110, M)
2-Cyanobenzyl alcohol (M)
Phthalide (111, M)
3-Cyanobenzyl alcohol (M)
4-Cyanobenzyl alcohol (95%,
2 days)
o-Nitrobenzyl alcohol (M)
m-Nitrobenzyl alcohol (M)
p-Nitrobenzyl alcohol (M)
o-Tolualcohol (M)
m-Tolualcohol (M)
m-Toluic acid (m)
p-Tolualcohol (M)
p-Toluic acid (m)
Phthalide (111, M)
Terephthalalcoholic acid (M)
Phthalidc (111, M)
Isophthalaldehydic alcohol (M)
Isophthalalcohol (M)
Terephthalaldehydic
alcohol (M)
Tcrcphthalalcohol (M)
0- Vanillyl alcohol (112, M)
3-Nitrosalicyl alcohol (113, M)
Vanillyl alcohol (114, M)
Veratryl alcohol (115, M)

205
240
190
200
190
90

Isovanillyl alcohol (116, M)


Ethylvanillyl alcohol (117, M)
2-Hydroxy-5-methoxybenzyl
alcohol (l1S, M)
2,4-Dimethylbenzyl alcohol
(100%, I day)

440 (2200) 242


120 (600) 171
40
130 (650)

30
30
60
30

170

10 30

190

20

80

101

30
20

40

82
141
265
110
60

10
20
40
40
30

60

20

234
265
36
600

132
21
242

287

222

120
34
240
400

(600)
20
(100)
21
(1200) 241
(2000) 233

50

20

26

20

130

31

204

y. Noma and Y. Asakawa

Table 2. Continued

3,4-Dihydroxybenzaldehyde (SO,
Protocatechualdehyde)
2,3-Dimethoxybenzaldehyde (Sl)

2,3-Dimethoxybenzyl
alcohol (100%, 1 day)
2,4-Dimethoxybenzaldehyde (S2) 2,4-Dimethoxybenzyl
alcohol (100%, 1 day)
2,5-Dimethoxybenzaldehyde (S3) 2,5-Dimethoxybenzyl
alcohol (100%, 1 day)
3,5-Dimethoxybenzaldehyde (S4) 3,5-Dimethoxybenzyl
alcohol (100%, 1 day)
2,3-Dichlorobenzyl
2,3-Dichlorobenzaldehyde (S5)
alcohol (100%, 1 day)
2,4-Dichlorobenzyl
2,4-Dichlorobenzaldehyde (S6)
alcohol (100%, 1 day)
2,6-Dichlorobenzaldehyde (S7)
2,6-Dichlorobenzyl
alcohol (100%, 1 day)
3,4-Dichlorobenzyl
3,4-Dichlorobenzaldehyde (SS)
alcohol (100%, 1 day)
3,5-Dichlorobenzyl
3,5-Dichlorobenzaldehyde (S9)
alcohol (100%, 1 day)
2,3,4-Trimethoxybenzaldehyde (90) 2,3,4-Trimethoxybenzyl
alcohol (100%, 1 day)
3,4,5-Trimethoxybenzaldehyde (91) 3,4,5-Dimethoxybenzyl
alcohol (100%, 1 day)
p-Acetylamidebenzaldehyde (92) p-Acetylamidebenzyl
alcohol (100%, 1 day)
tl-Phenylethanol (M)
Phenylacetaldehyde (93)
2-Phenylpropionaldehyde (94)
2-Phenylpropanol (M)
2-Phenylpropionic acid (m)
3-Phenylpropanol (120, M)
3-Phenylpropionaldehyde (95)
3-Phenylpropionic acid (m)
Cinnamyl alcohol (119, M)
Cinnamic aldehyde (96)
3-Phenylpropanol (120, M)
Cinnamic acid (m)
a-Methylcinnamic aldehyde (97)
2-Methylcinnamyl alcohol
(121, M)
2-Methyl-3-phenylpropanol
(122, M)
Nicotin aldehyde (9S)
Nicotin alcohol (M)
n-Heptylaldehyde (99, nn-Hexanol (100%, 2 day)
Enanthaldehyde, n- Heptanal)
Pelargonaldehyde (100,
n-Nonanol (41 %,2 days)
C9-aldehyde, Nonanal)
n-Nonanoic acid (41 %,2 days)
trans-2-Hexenal (101)
trans-2-Hexen-1-ol(60%,
1 day)
n-Hexanol (36%, 1 day)
trans-2-Heptenal (102)
trans-2-Hepten-1-ol (24%,
1 days)
n-Heptanol (19%, 1 day)
cis-4-Heptenal (103)
cis-4-Hepten-1-ol (82%, 3 days)
trans-2-Decenal (104)
trans-2-Decen-1-o1 (3%, 3 days)
n-Decanol (12%, 3 day)
2,4-Hexadienal (105)
2,4-Hexadien-l-01 (45%, 1 day)
4-Hexen-1-o1 (24%, 1 day)

1444

273

140

112

90

10

90

64

70

50

10
10
20
10
10
130

113

160

214

270

339

110
90

150
90

20

20

20

20

30

20

10
10

10

10

30

10

10

10

30
105

35

35

10

Table 2. Continued

trans,trans-2,4-Heptadien-l-01
(51 %,2 days)
trans-4-Hepten-l-01 (19%,
2, days)
trans,trans-2,4-Nonadien-l01 (34%, 1 day)
trans-4-Nonen-l-01 (66%, 1 day)

trans,trans-2,4-Heptadienal (106)

trans ,trans- 2,4- N onadienal (107)

10

65

10

" T.r. transformation ratio.


b D.C. lethal concentration of substrate for Euglena.
, M major product.
d m minor product.
, E.g. photo heterotrophic cultured Euglena.
f E.a. photo autotrophic cultured Euglena.
g E. w. heterotrophic cultured bleeched type Euglena.

2200

74(116)

2000

S..
:a
e
5

1500

0
0

on

bb

..

::c
'0
'0

"
~
~"

1000

..

..Q

rJJ

500
400
300
200
100
10

15

20

25

28

Time (days)

Fig. 4. Relationship between continuous addition of substrates, formation of metabolites, and


survival of E. gracilis Z. *The number in parentheses indicates the metabolite. #The concentration
of the substrates(70-76) causing death of E. gracilis Z. Compounds are as in Table 2. (Noma et al.
1991a)

206

Y Noma and Y Asakawa


ethylvanillin

vanillin
vanillyl alcohol(114)
100 .

light

ethylvanillyl alcohol(117)

100

light

80

80

'"
U

::I
't:O

...

dark

60

60

dark

40

40

20

20

O~--~--~----~--~--~

time(days)

OQC--~--~--------~--~

time(days)

Fig. 5. Effect of light illumination (ca. 3000 Ix) on the biotransformation of vanillin(72) and
ethylvanillin(75) by E. gracilis Z. (Y. Noma, unpubJ.)

The biotransformation of terpene ketones and related compounds by


Euglena is summarized in Table 3 (Noma 1987; Noma et al. 1989, 1992a,b,

1993, 1994a, 1995; Noma and Asakawa 1992). l-Carvone (123) was
stereospecifically transformed via d-dihydrocarvone (124) to dneodihydrocarveol (126), which was further transformed to 8-hydroxy-dneodihydrocarveol (130), namely (lR, 2S, 4R)-p-menthane-2,8-diol. l-n-,
d-iso-, and d-neoisodihydrocarveol (127-129) were also hydroxylated to give
8-hydroxydihydrocarveol (131-133), respectively (Fig. 6). On the other
hand, d-carvone (123') was also transformed mainly via l-isodihydrocarvone
(125') to l-isodihydrocarveol (128') as the major product and 1neoisodihydrocarveol (129'), together with l-dihydrocarvone (124') and 1neodihydrocarveol (126'), of which compounds 126', 128', and 129' were
further hydroxylated at the C-8 position to give the corresponding 8hydroxydihydrocarveols (130', 132', and 133'). d-Dihydrocarveol (127') was
also hydroxylated to give 8-hydroxy-d-dihydrocarveol (131') (Fig. 7). Hydrogenation of C = C double bond, reduction of C = 0 group and hydroxylation
at the C-8 position in the biotransformation of carvone occur under both light
and dark conditions. 1-Aminobenzotriazole used as a cytochrome P-450
inhibitor did not affect the inhibition of hydroxylation of dihydrocarveols
by Euglena (Noma et al. 1994a). l-Carvotanacetone (143) was also hydrogenated to d-neocarvomenthol (145) via d-carvomenthone (144). On the other
hand, the d-isomer (143') was also transformed to l-iso-(147) and 1neoisocarvomenthol (148) via l-isocarvomenthone (146) as the major products
(Fig. 8; Noma et al. 1989). (4R, 8RIS)-I-Carvone-8,9-epoxides (149a and 149b)
as the metabolites of l-carvone (123) by Streptomyces bottropensis, SY-2-1
(Noma and Nishimura 1982) were transformed via d-dihydrocarvone-8,9epoxides (150a and 150b) to d-neodihydrocarveol-8,9-epoxides (ISla and

Euglena gracilis Z

207

(y0R
/'"
137
138

(y0H
8a

&0

./

/'"
124

kI

-)I'

Ho"",6
./"
142

"6 ~

"""OH

/'"
135

/128
"

6" ,

&0 " cY"125

OR

Fig. 6. Biotransformation of l-carvone(123), l-trans-carveo!(134), 1-!imonene(I36) and related


compounds by Euglena gracilis Z. Compounds except 137-140 are as in Tab!es 1,3, and 6. 137 [trans-carvey! acetate; 138 I-trans-carvey! propionate; 139 I-cis-carvey! acetate; 140 [-cis-carvey!
propionate. (Noma 1987; Noma and Asakawa 1992; Noma et al. 1989, 1993, 1994a)

151b) as the major products together with d-isodihydrocarvone-8,9-epoxides,


n-l-, d-iso-, and d-neoisodihydrocarveol-8,9-epoxides as the minor products,
whereas d-isomers (149a' and 149b') were also transformed to 1isodihydrocarvone-8,9-epoxides (152a and 152b) as the major product and 1dihydrocarvone-8,9-epoxides (150a' and 150b') as the minor product. Further
reduction of 150a' and 150b' which gives the corresponding alcohols were
difficult. 2-Methyl-2-cyclohexenone (153) was easily transformed via 1-2methylcyclohexanone (154) to d-cis-2-methylcyclohexanol (155) as the major
products (Fig. 8; Noma et al. 1989). 2-Cyclohexenone (156) was strongly
phytotoxic at a concentration of lOOmg/l. However, compound 156 was easily
transformed to cyclohexanol (158) via cyclohexanone (157) as the major product. Further, dihydrocarvones (124, 125, 124', and 125'), carvomethones (144
and 146), dl-2-, 3-, and 4-methylcyclohexanones and cyclohexanone (157) as
saturated ketones were easily reduced to the corresponding alcohols.

208

y. Noma and Y. Asakawa

Table 3. Summary of biotransformation of mono terpene ketones and related compounds by


Euglena (Noma 1987; Noma and Asakawa 1992; Noma et al. 1989, 1990, 1993, 1994a, 1995)

Substrates
I-Carvone (123)
d-Carvone (123')

dl-Carvone (123 and 123')

l-Carvotanacetone (143)

d-Carvotanacetone (143')

dl-Carvotanacetone
(143 and 143')

I-Carvone-8,9-epoxides
(149a and b, 4R, 8RIS)

d-Carvone-8,9-epoxide (149a'
and b', 4S, 8RIS)
2-Methyl-2-cyc1ohexenone (153)
2-Cyclohexenone (156)
dl-Menthenone (159)
dl-Piperitone (160)
dl-Isopiperitenone (161)

Products [' T.r. (%)]


d-Dihydrocarvone (124, Mb )
d-Neodihydrocarveol (126, M)
d-8-Hydroxyneodihydrocarveol (130, M)
I-Dihydrocarvone (124', me)
l-Isodihydrocarvone (125', M)
I-Neodihydrocarveol (126', m)
1-Isodihydrocarveol (128', M)
I-Neoisodihydrocarveol (129', M)
1-8-Hydroxyneodihydrocarveol (130', M)
1-8-Hydroxyisodihydrocarveol (132', M)
1-8-Hydroxyneoisodihydrocarveol (133', M)
d-Dihydrocarvone (124, M)
d-Neodihydrocarveol (126, M)
I-Isodihydrocarvone (125', M)
1-Isodihydrocarveol (128', M)
I-Neoisodihydrocarveol (129', M)
d-8-Hydroxyneodihydrocarveol (130, M)
1-8-Hydroxyneodihydrocarveol (130', M)
1-8-Hydroxyisodihydrocarveol (132', M)
1-8-Hydroxyneoisodihydrocarveol (133', M)
d-Carvomenthone (144, M)
d-Isocarvomenthone (m)
d-Neocarvomenthol (145, M)
l-Carvomenthol(m)
I-Isocarvomenthone (146, M)
I-Carvomenthone (144', m)
l-Neocarvomenthol (m)
I-Isocarvomenthol (147, M)
l-Neoisocarvomenthol (148, M)
d-Carvomenthone (144, M)
l-Isocarvomenthone (146, M)
d-Neocarvomenthol (145, M)
l-Neoisocarvomenthol (148, M)
l-Isocarvomenthol (147, m)
l-Carvomenthol (m)
d-Dihydrocarvone-8,9-epoxides (150a and
b, 8RIS, M)
d-Isodihydrocarvone-8,9-epoxides (8RIS, m)
d-Neodihydrocarveol-8,9-epoxides
(ISla and b, 8RIS, M)
I-Dihydrocarveol-8,9-epoxides (8RIS, m)
d-Isodihydrocarveol-8,9-epoxides (8RIS, m)
d-Neoisodihydrocarveol-8,9-epoxides (8RIS, m)
I-Isodihydrocarvone-8,9-epoxides (152a and b, 8RIS, M)
I-Dihydrocarvone-8,9-epoxides (150a' and b', 8RIS, m)
1-2-Methy1cyclohexanone (154,3%,4 days, m)
d-cis-2-Methy1cyc1ohexanol (155, 88%, 4 days)
l-trans-2-Methy1cyclohexanol(7%, 4 days)
Cyclohexanone (157, 29%, 2 days)
Cyclohexanol (158, 67%, 2 days)
Not transformed
Not transformed
Not transformed

209

Euglena gracilis Z
Table 3. Continued

n-Piperitenone (162)
d-Pulegone (163)
d-Verbenone (164)
3-Methyl-2-cyclohexenone (165)
Isophorone(3,5,5-trimethyl-2cyclohexenone)
4-0xoisophorone
d-Dihydrocarvone and disodihydrocarvone (124 and
125, 80: 20 peak area in GC)

l-Dihydrocarvone and 1isodihydrocarvone (124' and


125', 80: 20 and 20: 80)

d- Dihydrocarvone-8, 9-epoxides
(150a and b)
l-Dihydrocarvone-8,9-epoxides
(150a' and b')
d-Carvomenthone (144)
l-Isocarvomenthone (146)

dl-Menthone
I-Camphor (250')
d-Camphor (250)
dl-Camphor (250 and 250')
dl-2-Methylcyclohexanone
dl-3-Methylcyclohexanone
dl-4- Methylcyclohexanone

Cyclobutanone
Cyclopentanone
Cyclohexanone (157)
Cycloheptanone
Cyclooctanone
Cyclodecanone
Cyclododecanone

, T.r. transformation ratio.


b M major product.
, m minor product.

Not
Not
Not
Not
Not

transformed
transformed
transformed
transformed
transformed

4-0xodihydroisophorone (M)
3,5,5-Trimethyl-4-oxocyclohexanol (M)
d-Neodihydrocarveol (126, M)
d-Isodihydrocarveol (128, m)
d-Neoisodihydrocarveol (129, m)
d-8-Hydroxyneodihydrocarveol (130, M)
d-8-Hydroxyisodihydrocarveol (132, M)
d-8-Hydroxyneoisodihydrocarveol (133, M)
l-Neodihydrocarveol (126', M)
l-Isodihydrocarveol (128', m)
I-Neoisodihydrocarveol (129', m)
1-8- Hydroxyneodihydrocarveol
(130, M)
1-8-Hydroxyisodihydrocarveol (132', M)
1-8-Hydroxyneoisodihydrocarveol (133', M)
d- Neodihydrocarveol-8, 9-epoxide
(151a, and b, M)
Not transformed
d-Neocarvomenthol (145, M)
d-Isocarvomenthol (147, m)
d-Neoisocarvomenthol (148, m)
Not transformed
d-Isoborneol (249', 22%, 14 days)
d-Borneol (248, 18%, 14 days)
d-Isoborneol (249', 16%, 14 days)
d-Borneol (248, 5%, 14 days)
cis- and trans-2-Methylcyclohexanol (46: 39,
peak area in GC)
cis- and trans-3-Methylcyclohexanol (80: 20,
peak area in GC)
cis- and trans-4-Methylcyclohexanol (25: 75,
peak area in GC)
Cyclobutanol(80%, 1 day)
Not transformed
Cyclohexanol (158, 100%, 11 days)
Cycloheptanol (76%, 6 days)
Cyclooctanol (38%, 6 days)
Cyclodecanol (16%, 10 days)
Cyclododecanol (83%, 10 days)

2,

142'

136'

2
,
\

./

123'

'~a

./
125'

~r"

137'

2/0R

134'

124'

2;OH * '" ~O

135'

qOH
129'

7" -

oJr-.

./

127'

~H

126'

130'

9~OH

132'

9:0H

133'

OH

131'

_ ~OH

~tH -

128'

" 2~H -

~OH

Fig. 7. Biotransformation of d-carvone(123'), d-cis-carveol(135'), d-limonene(I36'), and related compounds by Euglena


gracilis Z. Compounds except 137'-139' are as in Tables 1,3, and 6.137' d-trans-carveyl acetate; 139' d-cis-carveyl acetate.
(Noma 1987; Noma and Asakawa 1992, Noma et al. 1989, 1993, 1994a)

H01

141'

Ho/2 -

8b

CH 20H

139'

qOR
to

"""'

'"

V>

'"
::>
'0-"

Euglena gracilis Z

211

Oo~
-

(f0H ~' --~"

144

143

143'

145

146

2"'""

,/

147

'"

~OH
14S

0 0
0

(y0H

~
149a

0" 0

)<}

}<J

149b

150b

151b

oo~
153

0 ---

0 0 ---

154

157

~" ~o
159

160

/~~r *

8R

(y0H

8~

156

ISla

150a

~c

"-

149b'

2" *
150b'

(f0H
()OH
155

~'0

~c,~

152a

8S

149a'

ISS

"-

2;" *
150a'

~ ~o ~o
161

152b

162

163

~o
164

(\0
165

Fig.8. Biotransformation of carvotanacetone(143 and 143'), carvone-8,9-epoxide(149a, b, a' and


b'), 2-methyl-2-cyclohexenone(153) and 2-cyclohexenone(156) by Euglena gracilis Z. Compounds
are as in Table 3. (Noma et al. 1989)

Y. Noma and Y. Asakawa

212

Although men then one (159) was slightly reduced to menthone, piperitone
(160), isopiperitenone (161), n-piperitenone (162), pulegone (163), verbenone
(164), and 3-methyl-2-cyclohexenone (165) were not transformed at all (Fig. 8;
Noma et al. 1989). Although isophorone and dihydroisophorone were not
transformed at all, 4-oxoisophorone was easily transformed to 4oxodihydroisophorone and 3,3,5-trimethyl-4-oxocyclohexanol. The efficient
formation of saturated ketones such as 124, 125',144,146, 150a and 150b, 152a
and 152b, and 154 from the corresponding a,,B-unsaturated ketones (123, 123' ,
143, 143', 149a, 149b, 149a', 149b', and 153) suggested that the C = C double
bond may be hydrogenated from behind (Si-face), if the compounds possess-

OH
C6 re face
attack

H sisi-re
-'c
.,

..

re-re-si

H I-

z~

C6 si face

R'('R2

attack

I-Carvone(123)

Isopropenyl

d-Carvone(123')

I-Carvotanacelone(143)

Isopropyl

d-Carvotanacetone(143')

I-Carvone-8,9-epoxide
(1493 and b)

Epoxyisopropenyl

d-Carvone-8,9-epoxide
(1493' and b')

2-Methyl-2-cyclohexenone
(153)

C6 si face
attack

RI

C I si face and C Z
re face attack

Substrate

C6 re face
attack

Rz

Main Products

d-Dihydrocarvone(124)
d-Neodihydrocarveol(126)
Isopropenyl I-Isodihydrocarvone(125')
I-Isodihydrocarveol (128')
H
d-Carvomenthone(144)
d-Neocarvomenthol(145)
Isopropyl
1-Isocarvomenthone(146)
d-Neoisocarvomenthol(148)
H
d-Dihydrocarvone-8,9- epoxide(1503 and b)
d-Neodihydrocarveol-8,9-epoxide(1513 and b)
H

Epoxyisopropenyl
H

I-Isodihydrocarvone-8,9-epoxide(1523 and b)
1-2-Methylcyclohexanone(154)
d-2-Methylcyclohexanol(155)

Fig. 9. The stereospecific hydrogenation of the C = C double bond of a,!3-unsaturated ketones


and reduction of saturated ketones by Euglena gracilis Z. (Noma et al. 1989, 1995)

213

Euglena gracilis Z

ing 2-methyl-2-cyclohexenone skeleton are presented as shown in Fig. 9. The


stereospecific hydrogenation occurs independently on the configuration and
the kinds of the substituent at C-4 position, so that the methyl group at C-1
position is fixed mainly at R configuration. [2-2H]-I-Carvone ([2}H]-123) was
synthesized in order to clear up the hydrogenation mechanism at C-2 by
microorganisms. Compound [2-2H]-123 was also easily biotransformed to [22H]-8-hydroxy-d-neodihydrocarveol ([2-2H]-130) via [2-2H]-d-dihydrocarvone
([2-2H]-124) and [2- 2H]-d-neodihydrocarveol ([2- 2H]-126). On the basis of IH_
NMR spectral data of compound [2-2H]-130, we consider that the hydrogen
addition to the carbon-carbon double bond at the C j and C2 position by
Euglena occurred from the si face and re face, respectively, namely, anti
addition (Noma et al. 1995). On the other hand, the reduction of carbonyl
group in 124, 144, 146, 150a, 150b, and 154 occurs stereospecifically from the
Re face, except the predominant Si face attack for 125'. Although the resting
cell of Euglena also easily biotransformed 123' ,.143, 153, and 156, strong
inhibition was shown for the biotransformation of 123, dl-carvone (123 and
123'), 143', and dl-carvotanacetone (143 and 143'). The effect of substrate
concentration on the biotransformation by using 123 and 123' under light
illumination at ca. 3000lx was examined, and the result is shown in Fig. 10.
Euglena died at a concentration of 250 or 350mg/l and did not biotransform
carvone. On the basis of these results, we used the substrate at a concentration
of 100mg/l to carry out the biotransformation. The biotransformation of
123 by wild-type (E.g. and E.a.) and streptomycin bleached-type Euglena
(strain SM-ZK) cultured heterotrophically, photoheterotrophically, or
photoautotrophically was carried out with or without light illumination, and
the result is shown in Fig. 11. In the cases of photoheterotrophic and
(%)

100

123'

80

equal mixture of 123 and 123'


50

o~~~--~~~~~~~~~~

50 100 150 200 250 300 350 400 450 SOD (ppm)

Concentration
Fig. 10. Effect of carvone concentration(123, 123', and equal mixture of 123 and 123') on
biotransformation by E. gracilis Z. (Y. Noma, unpub!.)

214

y. Noma and Y. Asakawa


E.a.l.
E.g.!.

E.w.d

E.a.d.
E.g.d.

5
Time(days)

Time(days)

Time(days)

Fig. 11. Effect of light illumination on the biotransformation of l-carvone(123) in cases of


photoheterotrophical culture Euglena(E.g.), photoautotrophical culture Euglena(E.a.), and
bleached-type Euglena(E. w.): l, light; d. dark. (Noma et al. 1989)

photo autotrophic cultural Euglena, the velocity of biotransformation for 123


under light illumination was faster than that in the dark. However, in the case
of E. gracilis, strain SM-ZK biotransformation was carried out at nearly equal
speed under light and dark conditions. The time courses for the growth of
Euglena cultured photoheterotrophically (E.g.) in the presence of different
kinds of 123 concentration (50-400mgll, 50mg/1 interval) and the
biotransformation ratios are shown in Fig. 12. Growth was inhibited at a
concentration of 300mg/1 and upward of 123. However, on growing, Euglena
biotransformed 123 completely (Fig. 12). It is interesting to know what kinds
of light color are necessary for both the growth of Euglena and the
biotransformation of 123. As shown in Fig. 13, two kinds of light, namely, blue
and yellow to red colors, were efficient for the growth of Euglena and
biotransformation.
The formation of cyclohexanol (158) in the reduction of cyclohexanone
(157) as the metabolite of 2-cyclohexenone (156) by Euglena led our attention
to the biotransformations of dl-a- and f3-ionones (166 and 167), acetophenone
(174), and related compounds containing a,f3-unsaturated ketones and saturated ketones, cycloalkanones, and cycloalkanols (Fig. 8; Noma et al. 1989).
The results of the biotransformation of cycloalkanones (C4--C8, ClO, and C12)
and cycloalkanols (C4-C8, CI0, and C12) by Euglena are shown in Fig. 14
(Noma et al. 1990, 1992b). Euglena reduced cycloalkanones such as C4, C6C8, CI0, and C12 to the corresponding cycloalkanols. In the case of
cyclobutanone (C4), in the beginning, it was easily reduced to cyclobutanol,
which was gradually dehydrogenated to cyclobutanone again. The order of
preference for the reduction of cycloalkanones was C4 > C7 > C6 > C12 > C8
> CI0. On the other hand, the order of preference for the dehydrogenation
of the cycloalkanols was C5 > C4 > CI0 > C8 > C7 > C12. Cyclopentanone
was not reduced and cyclohexanol (158) was not dehydrogenated at all. In the
case of the biotransformations of an equal mixture of cycloalkanones and
cycloalkanols, the same phenomenon as described above was observed. When

/;r,oo-?--%

Time (days)

10

11

12

22

32

~.~rl~GOO~

"

"

'0

"e

01

50
~

.S
;;;

Concentration (ppm)

50 100 150 200 250 300 350 l.00

Fig. 12. Effect of l-carvone(123) concentration(50-400mg/l. 50mg/l interval) on the growth of E. gracilis Z and biotransformation. (Y. Noma.
unpub\.)

l.

'-~k:~~;/ 'i

5~~

:9

100

f-'

Uo

~
N

~.

f'"

216

y. Noma and Y. Asakawa

1.0
0.9

100

,...... 0.8

S
c:

'<t
\0

'i;:j

ci
0
;;::

0.7
0.6
0.5

(,)

0.4

...

0.3

<t:

0.2

.D
0
.D

'"

40

o--f5~
20

0.1
0

0
>.

OJ

e.
~

bJ)

:a

.s

..2
~

5 ..9~
... o:i
0
>~

'0

...
0

0<:

bJ)

u
'"'" 5
-;::: ;
~

..90.
o '"
u
!:l

Fig. 13. Effect of light color on growth and biotransformation of l-carvone(123) by Euglena
gracilis Z under light illumination at ca. 3000 Ix. (Y. Noma. unpubl.)

a mixture of cyc1opentanol and cyc1ohexanone (157) was added to the same


cultured broth at the same time, both dehydrogenation of cyc1opentanol and
reduction of 157 were carried out at the same time. Furthermore, in the
biotransformation of a mixture of cylcloalkanones (C6, C7, and C12) and
cycloalkanols (C4, CS, C8, and C10) at the same time the same phenomena
occurred (Fig. 15). Biotransformations of dl-a- and j3-ionones (166 and 167),
acetophenone (174), and related compounds containing a,j3-unsaturated
ketones and saturated ketones by Euglena were carried out with the substrate
concentration for the survival of Euglena (Table 4; Noma et al. 1992a,b).
Compounds 166 and 167 as norterpenes showed high toxity even at a low
concentration of ca. SOmg/l. However, compound 166 was biotransformed to
a-ionol and 7,8-dihydro-a-ionone as the major products and 7,8-dihydro-aionol as the minor product at a very low concentration, less than 40mg/1
(Noma et al. 1992b). On the other hand, compound 167 was biotransformed to
j3-ionol, 7,8-dihydro-j3-ionone, and 7,8-dihydro-j3-ionol. Benzalacetone (168)
was also transformed to 7,8-dihydrobenzalacetone and 4-phenyl-2-butanol.
Benzalacetophenone (chalcone, 169) was transformed to 7,8dihydrobenzalacetophenone (dihydrochalcone, which is known as a sweetener) and 1,3-diphenyl-2-propanol. Although 2-hydroxychalcone (170) was
not transformed at all, 2'-hydroxychalcone (171) was transformed to 2'hydroxy-7,8-dihydrochalcone in a low yield. 3-Hepten-2-one (172) was first
hydrogenated to 2-heptanone (232), which was easily reduced to 2-heptanol.

Euglena gracilis Z

217

8------

100

10--

50

50

7~
12

0
100

'"I:::
Q,)

8<

(TJ

~
0

50

(ij

100
0

~
50

()
>.

()
>.

100
5/

12

(ij

'"
"0

\00

15 (days)
0

y-l~
50

50

0,

,
2

,6

7_
12::::::0-..
,
,
100
5 6
7 (days)

Time

Fig. 14. Biotransformation of cycloalkanones and cycloalkanols by Euglena gracilis Z under light
illumination at ca. 3000 Ix. (Noma et al. 1992b )

A small amount of 2-butanone (229) and 2-butanol was formed at a low


concentration of 3-buten-2-one (173), which proved to be very toxic to
Euglena. However, compound 229, even at a high concentration, was easily
reduced to 2-butanol. As shown in Fig. 16, Euglena preferred the reduction of
the carbonyl group to the hydrogenation of the C = C double bond in the
biotransformations of 166 and 167. In the cases of 96 and 97, the reduction of
the aldehyde group occurs predominantly, and then the resulting alcohols (119
and 121) were further hydrogenated to the corresponding saturated alcohols
(120 and 122). However, in the cases of 168, 169, and 171-173, the hydrogenation of the C = C double bond in the side chain occurred predominantly
rather than the reduction of the carbonyl group (Fig. 16). Acetophenone
(174), m-hydroxy-(177) 0-, m-, and p-methoxy-(179, 181, and 183) and 0-,
m-, and p-methyl-(185, 187, and 189), p-heptyl-(191), 3,4-dimethoxy(192), 2,5-dimethyl-(193), 4-hydroxy-3-methoxy-(198, acetovanillone), 3,4,5-

""'

-0

-0

::c
o ,....

10

......

12
14
Ti me (m ; n. )

,co

o....;

::c
o

1~

.0

U
~

18

20

CsC=O

CsOH

22

ClOC=O

24

Fig. 15. Gas chromatogram for biotransformation of cycloalkanones(C6, C7, and C12) and cycloalkanols(C4, C5, C8, and CIO) mixture by Euglena gracilis
Z under light illumination at ca. 3000lx. (Y. Noma, unpubl.)

u
..;t
u

u
u

::c

u ,....

::c
o CO

;I>

p..

po

N
.....
00

p-Methoxyacetophenone (183)

m-Methoxyacetophenone (181)

o-Hydroxyacetophenone (176)
m-Hydroxyacetophenone (177)
p-Hydroxyacetophenone (178)
o-Methoxyacetophenone (179)

Acetophenone (174)

a-Methylcinnamic aldehyde (97)

3-Buten-2-one (173, methyl


vinyl ketone)
Cinnamic aldehyde (96)

2-Hydroxychalcone (170)
2' -Hydroxychalcone (171)
3-Hepten-2-one (172)

Benzalacetophenone (169, chalcone)

Benzalacetone (168)

f3-Ionone (167)

Substrates
dl-a-Ionone (166)

bD.C. (ul or mg/lOOml)


E.g.
E.a.
Products [" T.r.(%)J
a-Ionol (M')
5
50
7,8-Dihydro-a-ionone (M)
7,8-Dihydro-a-ionol (m d )
50
f3- lonol (M)
5
7,8-Dihydro-f3-ionone (M)
7,8-Dihydro-f3-ionol (m)
7,8-Dihydrobenzalacetone (4-Phenyl-2-butanone. 51 %,3 days)
75
4-Phenyl-2-butanol(49%, 3 days [aJI} = -8.5 (C = 004, CHCI,
30mg/400ml
7.8-Dihydrobenzalacetophenone (Dihydrochalcone. M)
156
1,3-Diphenyl-2-propanol (M)
Not transformed
30
40
2'-Hydroxydihydrochalcone (20%.4 days)
2-Heptanone (232, 80 and 68%, 1 and 2 days)
50
2-Heptanol (20 and 32%, 1 and 2 days)
2-Butanone (229, 93%, I day)
10
2-Butanol (7%, 1 day)
10
10
Cinnamyl alcohol (119, M)
3-Phenylpropanol (120, M)
10
10
2-Methylcinnamyl alcohol (121, M)
2-Methyl-3-phenyl-l-propanol (122, M)
I-Phenyl-l-ethanol (17Sa(R) and b(S), 100%. 6 days,
130
70
R:S = 25:75 and 45:55,16 and 44 days, respectively)
40
Not transformed
30
43
1-(m-Hydroxyphenyl)-I-ethanol (100%, 18 days, [aJI) = -34)
70
40
30
Not transformed
50
50
1-(o-Methoxyphenyl)-I-ethanol (180a(R) and b(S), 100%,
R:S = 50:50, 12 days)
50
1-(m-Methyoxyphenyl)-I-ethanol (182a(R) and b(S), 100%,5 days, 60
R:S = 32:68 and 50:50, 5 days and 27 days, respectively)
40
40
I-(p-Methyoxyphenyl)-I-ethanol (184a(R) and b(S), 67%,
14 days, R:S = 50:50)
67

E.w.l.

Table 4. Summary of biotransformation of a- and f3-ionones, acetophenone, and related compounds by Euglena. (Noma et al. 1992a, 1994b)

67

E.w.d

~
N

~Q.

i"

t>J

Isobutyrophenone (214)
Valerophenone (215, nPentanophenone)

o-Hydroxypropiophenone (210)
p-Hydroxypropiophenone (211)
n-Butyrophenone (212)

2-Hydroxy-5-methylacetophenone (195)
2-Hydroxy-4-methoxyacetophenone (196)
2-Hydroxy-5-methoxyacetophenone (197)
4-Hydroxy-3-methoxyacetophenone
(198, Acetovanillone)
4-Hydroxy-3-methylacetophenone (199)
2,4-Dihydroxyacetophenone (200)
2,5-Dihydroxyacetophenone (201)
2,6-Dihydroxyacetophenone (202)
3,4-Dihydroxyacetophenone (203)
3,5-Dihydroxyacetophenone (204)
2,4,6-Trihydroxyacetophenone (205)
3,4,5-Trimethoxyacetophenone (206)
2,4,6-Trimethylacetophenone (207)
Propiophenone (208)

p-n-Heptylacetophenone (191)
3,4-Dimethoxyacetophenone (192)
2,5-Dimethylacetophenone (193)

p-Methylacetophenone (189)

rn-Methylacetophenone (187)

o-Methylacetophenone (185)

Table 4. Continued

Not transformed
Not transformed
Not transformed
Not transformed
Not transformed
Not transformed
Not transformed
1-(3,4,5-Trimethoxyphenyl)-l-ethanol (10%, 3 days)
Not transformed
I-Phenyl-l-propanol (209a(R) and b(S), 100%,
7 days, R:S = 21 :79, [al D = -28.2)
Not transformed
Not transformed
I-Phenyl-l-butanol (213a(R) and b(S), 94%,
5 days, R:S = 42:58, [al D = -7, (16%ee
I-Phenyl-l-isobutanol (86%, 10 days)
I-Phenyl-l-pentanol (33%, 2 days, dead)

1-(o-Methylphenyl)-I-ethanol (l86a(R) and b(S), 96%,


16 days, R:S = 40:56)
1-(rn-Methylphenyl)-l-ethanol (l88a(R) and b(S),
95%,6 days, R:S = 44:56)
1-(p-Methylphenyl)-l-ethanol (l90a(R) and b(S), 85%, 5 days,
R:S = 20:80,49:51,4 and 25 days, respectively)
1-(p-n-Heptylphenyl)-l-ethanol (29%, 8 days, dead)
1-(3,4-Dimethoxyphenyl)-I-ethanol (55%, 14 days)
1-(2,5-Dimethylphenyl)-1-ethanol (194a(R) and b(S), 68%,
14 days, R:S = 46:54)
Not transformed
Not transformed
Not transformed
1-(4-Hydroxy-3-methoxyphenyl)-I-ethanol (58%, 14 days)

20
10

30
20
40

20
20
10
10
156
20
10
10
30

11

10
10
10
50

20
50
30

60

50

10

10
10
30

20

40

40
30

40

III

::;!

III

III

'"

~
)-

p,.

III
III

tv
tv
0

2-Aeetylcyclopentanone (228)
2-Butanone (229)
2-Pentanone (230, Methyl-npropyl ketone)
2-Hexanone (231, Methyl-nbutyl ketone)
2-Heptanone (232, Methyl-npentyl ketone)
2-0etanone (233, Methyl-nhexyl ketone)
2-Nonanone (234, Methyl-nheptyl ketone)
2-Deeanone (235, Methyl-noetyl ketone)
2-Undecanone (236, Methyl-nnoryl ketone)
2-Dodeeanone (237, Methyl-ndeeanyl ketone)
4-Methyl-2-pentanone (238)
Ethyl-n-butyl ketone (239)

2-Aeetylcylcohexanone (227)

n-Hexanophenone (216)
n-Heptanophenone (217)
p-Hydroxy-n-heptanophenone (218)
n-Octanophenone (219)
n-Nonanophenone (220)
n-Deeanophenone (221)
4-(p-Hydroxyphenyl)-2-butanone (222)
I-Phenyl-3-hydroxy-5-hexanone (223)
4-Phenyl-3-butanone (224,
I-Phenyl-2-butanone)
5-Aeetyl-2-norbornene (225)
Benzoyl acetone (226)

20
11O/500ml

2-Deeanol (82%, 4 days)

2-Undeeanol (91 %,5 days)

4-Methyl-2-pcntanol (40%, 6 days)


3-Heptanol (86%, 4 days)

930

20

2-Nonanol (94%)

2-Dodecanol (88%, 6 days)

170

560

10

10

80

10
10
10
10
20
40
110
33

2-0etanol (94%, 5 days)

2-Heptanol (83%,6 days)

2-Heptanol (58%,5 days)

5-(2' -Hydroxyethyl)-2-norbornene (28%, 3 days, dead)


4-Phenyl-4-hydroxy-2-butanone (39%, 4 days)
4-Phenyl-2-hydroxy-4-butanone(24%, 4 days)
4-Phenylbutane-2,4-diol (38%, 4 days)
2-Acetylcyclohexanol (34%, 3 days, dead)
2-(2' -Hydroxyethyl)-eyclohexanone (46%, 3 days, dead)
2-(2' -Hydroxyethyl)-cyclopentanone (25%, 3 days, dead)
2-Butanol 34%, 5 days)
2-Pentanol (29%,5 days)

4-(p-Hydroxyphenyl)-2-butanol
l-Phenylhexa-3,5-diol
4-Phenyl-3-butanol (100%, 4 days)

l-Phenyl-l-hexanol (72%, 4 days, dead)


l-Phenyl-l-heptanol (68%, 8 days, dead)
Not transformed
l-Phenyl-l-oetanol (44%, 4 days, dead)
l-Phenyl-l-nonanol (18%, 2 days, dead)

10

20

:::l.
~
N

:l

<>to

;:

"'"

tl']
;::
<>to

4-Heptanol (98%, 5 days)


Isomerization was occured each other
2-Hydroxy-3-pentanone (100%, 5 days)
Not transformed
5-Methyl-3-heptanol (71 %,5 days)
Yashabushidiol A
Yashabushidiol B
Benzhydrol (87%, 4 days)
3,3-Dimethyl-2-butanol (100%)
3,3,5-Trimethyl-4-oxocyclohexanol (M)

" T.r. transformation ratio.


b D.C. lethal substrate concentration for Euglena.
, M major product.
d m minor product.

Benzophenone(246)
3,3-Dimethyl-2-butanone (247, Pinacolin)
4-0xodihydroisophorone

Di-n-propyl ketone (240)


Geranylacetone and nerylacetone (241)
Acetylacetone (242, 2,4-Pentadione)
Acetonylacetone (243, 2,5-Hexadione)
5-Methyl-3-heptanone (244)
Dihydroyashabushiketol (245)

Table 4. Continued

1920

160
260
760
460

810

"''~""
'"

0-

'"

Z
o

!::S
tv

Euglena gracilis Z

223

OH

RI~R3

R2

Rl~R3
R2

Substrates

Rl

R2

R3

a-Ionone(166)

Me

~- lonone(167)

Me

Benzaldehyde(168)

Me

Benzalacetophenone(169)

2-Hydroxychalcone(170)

Cl

2'-Hydroxychalcone(171 )

"JGJ"

Cinnamic aldehyde(96)

a-Methylcinnamic
aldchyde(97)
3- Hepten-2 -one(172)

Me

C)H7

Me

3-Buten-2-one( 173)

Me

....

Oll

Products
a-Ionol
7,8-Dihydro-a-ionol
7,8-Dihydro-a-ionone
p-Ionol
7,8-Dihydro-p-ionol
7 ,8-Dihydro-~-ionone
7,8-Di hydrobenzalacetone
4-Phenyl-2-butanone
7,8- Dihydrobenzalacetophenone
1,3-Diphenylpropanol
Not transformed
2'-Hydroxydihydrochalcone
Cinnamyl alcohol(119)
3-Phenylpropanol( 120)
2-Methylcinnamyl alcohol(121)
2-Methyl-3-phenylpropanol( 122)
2 -Heptanone(232)
2-Heptanol
2-Butanone (229)
2-Butanol

Fig. 16. Metabolic pathways of a-(I66) and f3-ionones(167) and related compounds by Euglena
gracilis Z, (Noma et aL 1992b)

trimethoxyacetophenones (206) were also reduced to the corresponding secondary alcohols. The order of preference for the reduction was 181 > 187 >
174> 189 > 179 > 185 > 191 > 177 > 183 > 192 = 193> 198. lIowever,oand p-hydroxyacetophenone (176 and 178), 2-hydroxy-5-methyl-, 2-hydroxy-

224

y. Noma and Y. Asakawa

4-methoxy- and 2-hydroxy-5-methoxyacetophenones (195-197), 4-hydroxy-3methyl-, 2,4-, 2,5-, 2,6-, 3,4-, and 3,5-dihydroxyacetophenones (199-204), and
2,4,6-trihydroxy-, and 2,4,6-trimethylacetophenones (205 and 207) were not
transformed at all (Fig. 17; Noma et al. 1994b). Enanti()selectivity in the
biotransformation of compounds 174, 177, 179-181, 189, 193 and 208 by
Euglena was investigated using an optical isomer separating capillary column
(CDX-B, j3-cyclodextrin); the result is shown in Fig. 18. In the case of compound 174, first (lS)-1-phenyl-1-ethanol (175b) was predominantly formed [RI
S ratio = 25:75; 50% enantiomer excess(ee)]. However, with time, (lR-)l-

Fig. 17. Biotransformation of acetophenone(174) and related compounds by Euglena gracilis Z.


Compounds as in Table 4. (Noma et al. 1994b)

225

Euglena gracilis Z

Time (days)

"

(l~
O:vte

179
.

~
~

en

/~

ti

::s

'd

....0

,,0

/~

/~----- - - j\ - - - -..1-

llll)

jY/

~en
180b --'"
<~

/4~.~~-

0..

I ~2b

182a and b

180a and b /' ~~

nHl

ti

::s

'd

....

- jf .

'iO

_1r-

0..

;t,

-'

O.j<C-~~~-~~---~~----~~J~
11

'6

Time (days)

10

I)

V __ ~_~-Q--~~--.--!,\~~

1112),5678

Time (days)

27

209a and b
IUU

189

~
en

ti
::s

'd
0

....

00

0..

Time (days)

10

Time (days)

Fig. 18. Enantioselectivity in the biotransformation of acetophenone(174), ()- and mmethoxyacetophenones(179 and 181), p-methylacetophenone(189), and n-propiophenone(208)
by Euglena gracilis Z. Compounds as in Table 4. (Noma et al. 1994b)

226

y. Noma and Y. Asakawa

phenyl-I-ethanol (175a) gradually increased, and finally the RIS ratio became
45:55 (10% ee) (Noma et al. 1994b). Compounds 175a and 175b were
dehydrogenated to give 174 in a small amount. In the case of compound 179,
the RIS ratio of 180a and 180b was nearly equal. In compound 181, when the
biotransformation was finished, the RIS ratio of 182a and 182b changed to
32:68 (36% ee). However, when the cultured medium including 181 was
allowed to stand for a long time (22 days) at room temperature, the RIS ratio
reached equal again. In compound 189, although the RIS ratio of 190a and
190b became 20: 47 (27% ee) in the course of the reaction, it was equal by the
end. Propiophenone (208) was transformed predominantly to (IS)-I-phenyl-lpropanols (209b, 59% ee). n-Butyrophenone (212) was transformed predominantly to (IS)-I-phenyl-l-butanol (213b, 16% ee). The results of the
biotransformations of n-propio-, n-butyro-, isobutyro-, n-heptano-, n-octano-,
n-nonano-, and n-decanophenones (187-193) and the substrate concentration
of the survival of Euglena are shown in Fig. 17 and Table 4 (Noma et al. 1992a,
1994b). Compounds 187-192 were also transformed to the corresponding
secondary alcohols. However, 0- and p-hydroxypropiophenones (210 and 211)
and p-hydroxyheptanophenone (218) were not transformed at all (Table 4). In
the biotransformation of aliphatic methyl ketones such as 2-butanone (229),2pentanone (230), 2-hexanone (231), 2-heptanone (232), 2-octanone (233), 2nonanone (234), 2-decanone (235), 2-undecanone (236), and 2-dodecanone
(237), all compounds were reduced to the corresponding alcohols and the
order of preference for the reduction was 235 > 234 > 236 > 233 > 237 > 232
> 231 > 229 > 230 (Fig. 19). On the basis of the above results, it is suggested
that the longer side chain of aliphatic methyl ketones (229-237) increases
reactivity for the reduction of carbonyl group. 4-Methyl-2-pentanone (238),
ethyl-n-butyl ketone (239), di-n-propyl ketone (240), acetyl acetone (242), 5methyl-3-heptanone (244), and 4-(p-hydroxyphenyl)-2-butanone (222), 1phenyl-2-butanone (224), pinacolin (247, 3,3-dimethyl-2-butanone), and
benzophenone (246) were also transformed easily to the corresponding
alcohols. Further, benzoylacetone (226) was transformed to 4-phenyl-4hydroxy-2-butanone, 4-phenyl-2-hydroxy-4-butanone, and 4-phenylbutane2,4-diol. However, acetonylacetone (243) was not transformed at all.
Biotransformations of terpene alcohols and related alcohols by Euglena
are summarized in Table 5 (Noma et al. 1990, 1991b, 1992b, 1993, 1994a; Noma
and Asakawa 1992). The time courses and metabolic pathways for the
biotransformations of l-trans- and cis-carveols (134 and 135), d-trans- and ciscarveols (134' and 135') and dl-trans-carveol (equal mixture of 134 and 134')
and dl-cis-carveol (equal mixture of 135 and 135') by E. gracilis Z are shown
in Figs. 6, 7, and 20. Compound 134 was transformed stereospecifically via
123 and 124 to 126 as the major product. However, compound 135 was not
transformed (Figs. 6, 20). On the other hand, compound 135' was
diastereoselectively transformed, mainly to give 123', 125', and 128' as major
product, and compound 134' was not transformed at all (Figs. 7, 20).
Enantioselectivities for an equal mixture of 134 and 134' and 135 and 135' and
the diastereoselectivities for the mixture of 134 and 135 and 134' and 135' were
observed (Noma and Asakawa 1992). Furthermore, in the biotransformation

Euglena gracilis Z

227

100

50

2345678

Time(days)
Fig. 19. Biotransformation of aliphatic ketones(229-237) by Euglena gracilis Z. Compounds as in
Table 4. (Noma et al. 1994b)

of l-trans- and cis-carveyl acetates (137 and 139) and d-trans- and cis-carveyl
acetates (137' and 139') and I-trans- and cis-carveyl propionates (138 and 140),
Euglena easily hydrolyzed the esters to give 134, 135, 134' , and 135'. Although
the alcohols 134' and 135 were not transformed at all and accumulated in the
medium, compounds 134 and 135' were further metabolized to carvones (123
and 123') and other related metabolites as described above (Figs. 6, 7). The
preferential hydrolyses for cis-forms were observed. Furthermore, in the
biotransformations of d-, 1-, and dl-borneols (248, 248', and equal mixture of
248 and 248') and d-, 1-, and dl-isoborneols (249',249, and equal mixture of 249'
and 249), the enantio- and diastereoselective dehydrogenation for 249' was
observed and I-camphor (250') was obtained at ca. 50% yield (Fig. 21; Noma
et al. 1992b). The conversion ratio was ca. 50% even at the different kind of
concentration of 249' (Fig. 22). When compound 250' was used as a substrate,
it was also converted to 249' in 22% yield for 14 days. Furthermore, compound
d-camphor (250) was also reduced to 248 in 4 and 18% yield for 7 and 14 days,
respectively. Cycloalkanols such as cyclobutanol, cyclopentanol, cyclooctanol,
and cyclodecanol were easily dehydrogenated to the corresponding ketones, as
described above. The order of preference for the dehydrogenation of the
cycloalkanols was C5 > C4 > ClO > C8 > C7 > C12 (Fig. 14, Table 5).

228

y. Noma and Y. Asakawa

Table 5. Summary of biotransformation of terpene alcohols and related compounds by Euglena.


(Noma and Asakawa 1992. Noma et at. 1990, 1992b, 1993, 1994a)

Substrates
I-trans-Carveol (134)

I-cis-Carveol (135)
l-trans-Carveyl propionate (13S)
d-trans-Carveol (134')
d-cis-Carveol (135')

dl-trans-Carveol (134 and 134')

dl-cis-Carveol (135 and 135')

The mixture of I-trans- and


cis-Carveol (134 and 135)
The mixture of d-trans- and
cis-Carveol (134', and 135')
d-trans-Carveyl acetate (137')
l-cis-Carveyl acetate (139)
d-cis-Carveyl acetate (139')

l-cis-Carveyl propionate (140)


The mixture of (2S)-[2- 2H)-d-trans-Carveol
and (2S)-[2}H)-d-cis-Carveol
d-Neodihydrocarveol (U6)
I-Neodihydrocarveol (126')
d-Dihydrocarveol (127')
I-Dihydrocarveol (U7)
d- Isodihydrocarveol (12S)
I-Isodihydrocarveol (12S')
d-Neoisodihydrocarveol (129)
l-Neoisodihydrocarveol (U9')
l-Perillyl alcohol (Sa)

dl-Perillyl alcohol (Sa and Sb)

Products [a T.r. (%)


I-Carvone (U3, Mb)
d-Dihydrocarvone (124)
d-Neodihydrocarveol (U6, M)
d-8-Hydroxyneodihydrocarveol (130, me)
Not transformed
I-trans-Carveol (134, M)
d-Neodihydrocarveol (126, m)
Not transformed
d-Carvone (123', M)
I-Isodihydrocarvone (125', M)
l-Isodihydrocarveol (US', M)
l-Neoisodihydrocarveol (U9', M)
1-8-Hydroxyisodihydrocarveol (132', m)
1-8-Hydroxyneoisodihydrocarveol (133', m)
l-Carvone (U3, M)
d-Dihydrocarvone (U4, M)
d-Neodihydrocarveol (U6, M)
d-Carvone (123', M)
l-Isodihydrocarvone (US', M)
I-Isodihydrocarveol (US', M)
l-Neoisodihydrocarveol (U9', M)
I-Carvone (U3, M)
d-Dihydrocarvone (124, M)
d-Neodihydrocarveol (126, M)
d-8-Hydroxyneodihydrocarveol (130, m)
d-Carvone (123', M)
I-Isodihydrocarvone (US', M)
l-Isodihydrocarveol (US', M)
I-Neoisodihydrocarveol (U9', M)
d-trans-Carveol (134', M)
I-cis-Carveol (135, M)
d-cis-Carveol (135', M)
l-Neodihydrocarveol (126', M)
I-Isodihydrocarveol (US', M)
I-cis-Carveol (135, M)
d-Carvone (123', m)
I-Isodihydrocarvone (US', m)
d- Isodihydrocarveol (US', M)
d-8-Hydroxyneodihydrocarveol (130, M)
1-8-Hydroxyneodihydrocarveol (130', M)
d-8-Hydroxydihydrocarveol (131', M)
1-8-Hydroxydihydrocarveol (131, M)
d-8-Hydroxyisodihydrocarveol (132, M)
1-8-Hydroxyisodihydrocarveol (132', M)
d-8-Hydroxyneoisodihydrocarveol (133, M)
1-8-Hydroxyneoisodihydrocarveol (133', M)
trans-Shisool (9, M)
cis-Shisool (10, m)
I-Perillic acid (13a, m)
8-Hydroxy-trans-shisool (11, M)
trans-Shisool (9, M)
cis-Shisool (10, m)
dl-Perillic acid (13a and 13b, m)

229

Euglena gracilis Z

Table 5. Continued
The mixture of trans- and cis-Shisools
(9: 10 = 62: 38 peak area in GC)
l-Phellandrol (18)
Geraniol (32)
Nerol (33)
Geranyl acetate
d-Borneol (248)
I-Borneol (248')
dl-Borneol (248 and 248')
1- Isoborneol (249)
d-Isoborneol (249')
dl- Isoborneol (249' and 249)
Myrtenol (2)
Myrtanol (4a and b)
Thymol
Cumin alcohol (28)
l-Acetoxy-p-menthane
Cinnamyl alcohol (119)
2-Methylcinnamyl alcohol (121)
Eugenol
(lR)-l-Phenyl-l-ethanol (175a, R:S = 100:0)
(IS)-I-Phenyl-l-ethanol (175b, R:S
dl-l-Phenyl-l-ethanol (R:S

0:100)

50:50)

I-Buten-3-ol
2-Methylcyclohexanol(trans and cis

Cyclobutanol
Cyclopentanol
Cyclohexanol (158)
Cycioheptanol
Cyclooctanol
Cyclodecanol
Cyclododecanol

20: 80)

trans-Shisoic acid (16, M)


8-Hydroxy-trans-shisool (11, M)
8-Hydroxy-cis-shisool (12, M)
trans-Tetrahydroperillyl alcohol (21, m)
cis-Tetrahydroperillyl alcohol (22, m)
Nerol (33, M)
Citronellol (34 or 35, M)
Geraniol (32, M)
Citronellol (34 or 35, m)
Geraniol (32, M)
Nerol (33, M)
Not transformed
Not transformed
Not transformed
Not transformed
I-Camphor (250', ca, 50%)
I-Camphor (250', ca, 25 %)
Not transformed
Not transformed
Not transformed
Not transformed
p-Menthan-l-ol (65%, 5 days)
3-Phenylpropanol (120, M)
2-Methyl-3-phenylpropanol (122, M)
Not transformed
Acetophenone (174, m)
I-Phenyl-l-ethanol (R:S = 93:7,25 days)
Acetophenone (174,3%,25 days)
I-Phenyl-l-ethanol (R:S = 11 :87, 25 days)
Acetophenone (174, 2%, 25 days)
I-Phenyl-l-ethanol (R:S = 54:45,25 days)
I-Buten-3-one
2-Butanone
2-Butanol
2-Methylcyclohexanol (trans & cis = 15 :85)
2-Methylcyciohexanone
Cyclobutanone (98%,3 days)
Cyciopentanone (100%, 3 days)
Not transformed
Cycloheptanone (20%, 3 days)
Cyclooctanone (70%, 11 days)
Cyclodecanone (100%, 16 days)
Cyclododecanone (6%,6 days)

Tr. transformation ratio.


M major product.
, m minor product.
o

Cinnamyl alcohol (119) and 2-methylcinnamyl alcohol (121) were hydrogenated to 3-phenylpropanol (120) and 2-methyl-3-phenylpropanol (122), respectively (Table 5). When either (1R)- or (lS)-l-phenyl-l-ethanol (175a or 175b)
was transformed, acetophenone (174) was obtained in a small amount and the
RIS ratio of 175a and 175b became 93: 7 and 11: 87, respectively (Table 5).

230

Y. Noma and Y. Asakawa

135

100

134

~
:!l
u

50

::l

-0

123
0

I)

(,

126
1() II

Time (days)

::l

I)

Time (days)

135'

~
-0

134'

I(X)

:!l
u

Ii

50

0..

129'

I::=._.-----L-.
..
2
4
6 8
8-----8

10

10

Time (days)

Time (days)

100

134 and 134'

~
:!l
u
::l

-0

::
0..

50

L-..L

10 12 14 16 0

Time (days)

10 II

Time (days)

Fig. 20. Enantioselectivity in biotransformation of l-trans- and cis-carveol (134 and 135), d-transand cis-carveol(l34' and 135'), and dl-trans- and cis-carveol(l34 and 134' and 135 and 135') by
Euglena gracilis Z. Compounds as in Table 3 and Figs. 9 and 10. (Noma and Asakawa 1992)

1- Acetoxy-p-menthane and geranyl acetate were also hydrolyzed to pmenthan-1-01 and geraniol (30), respectively, as well as carveyl acetates,
carveyl propionate, etc. Compound 30 was isomerized to nerol (31). Terpene
alcohols containing isopropenyl group such as dihydrocarveol and shisool

----

O~

50

,
8

"',:,u

Time(days)

/'

-"""""""M"'.:

12
0

~,

,
4

8
Time(days)

'].."J~

"-

~<49'

248'

12

250'

8
Time(days)

fa 249 and 249'

12

Fig. 21. Enantio- and diastereoselectivity in the biotransformation of borneols by Euglena gracilis Z. Compounds: 248' I-borneol;
249 and 249' 1- and d-isoborneol, respectively; 250' I-camphor. (Noma et al. 1992b)

Q..

L..

"0

=>

tl

249

100~

f-'

'"

(")

;:,

232

Y. Noma and Y. Asakawa

,OH

100

249'

:.~ 100ppm

~~~~.

---0-.
~

'-'

"011)
E
0
..0
0

10
50

til
......

"I!:s

---Q

Irl
~

....0

'-'

..c:::

50

0..

-.!.

100
Time(days)
Fig. 22. Effect of substrate concentration on biotransformation of d-isoborneol by Euglena
gracilis Z. Compounds: 249' d-isoborneol; 250' I-camphor. (Y. Noma et at. 1992b)

were hydroxylated at the C-8 position to give to 8-hydroxy compounds (Figs.


6, 7; Noma et al. 1993, 1994a). 1-Aminobenzotriazole as cytochrome P-450
inhibitor did not inhibit the hydroxylations of dihydrocarveols and shisools
(Noma et al. 1993, 1994a).
The results of the biotransformations of terpene hydrocarbons and related
compounds are summarized in Table 6.I-Limonene (136) was biotransformed
to I-trans and cis-carveols (134 and 135), l-perilly alcohol (8a) and I-trans- and
cis-isopiperitenols (141 and 142) (Fig. 6). On the other hand, d-limonene
(136') was biotransformed to d-trans- and cis-carveols (134' and 135'), dperillyl alcohol (8b), and d-trans- and cis-isopiperitenols (141' and 142') (Fig.
7). 1-Methyl-1-cyclohexene and cyclohexene were transformed to 3-methyl-2cyclohexenone (165) and 2-cyclohexenol, respectively. However, 1,4- and 1,8cineoles, p-menthane, and /).3 -carene were not transformed at all.
We consider that the utilization of Euglena as a bioreactor is very interesting to prepare primary alcohol in the case of the biotransformation of aldehydes, very important fragrant components of which, such as shisool, etc. have
been obtained, and the enantio- and diastereoselective formation of secondary
alcohols in biotransformation of trans- and cis-carveols and borneols and
isoborneols and the selective formation of important terpene diol such as 8hydroxydihydrocarveol from carvone, dihydrocarvone, and dihydrocarveol.
Utilization of Euglena as a bioreactor is very convenient and useful, because
this method is one pot, has a short reaction time, and preparation is very
simple.

233

Euglena gracilis Z

Table 6. Summary of biotransformation of terpene hydrocarbon


and related compounds by Euglena. (Y. Noma, unpubl.)

Substrate
d-Limonene (136')

/-Limonene (136)

I-Methyl-l-cydohexene
Cyclohexene
l,4-Cineole
1,8-Cineole
~]-Carene

p-Menthane

Products
d-trans-Isopiperitenol (141')
d-cis-Isopiperitenol (142')
d-trans-Carveol (134')
d-cis-Carveol (135')
d- Perillyl alcohol (Sb)
d-Carvone (123')
I-Neodihydrocarveol (126')
/-trans- Isopiperitenol (141)
/-cis- Isopiperitenol (142)
/-trans-Carveol (134)
/-cis-Carveol (135)
I-Carvone (123)
/-Perillyl alcohol (Sa)
d-Neodihydrocarveol (126)
d-Isodihydrocarveol (12S)
3-Methyl-2-cydohexenone (165)
2-Cyclohexenol
Cyclohexanol (ISS)
Not transformed
Not transformed
Not transformed
Not transformed

4 Summary and Conclusion


The biotransformations of terpene aldehydes, terpene ketones, terpene
alcohols, terpene hydrocarbons, and the related compounds by Euglena
gracilis Z were carried out with the substrate concentration for the survival of
the phytoflagellate. The biotransformations were usually carried out at ca.
lOOmg/1 at first, because Euglena died at a substrate concentration above
200mg/l and did not biotransform the substrates. Most terpene aldehydes,
aromatic aldehydes, aliphatic aldehydes, and the related aldehydes were
biotransformed to the corresponding primary alcohols. However, some of
them gave the corresponding acids as major or minor products. a,{3Unsaturated terpene aldehydes were firstly reduced to give the corresponding
alcohols, which were further hydrogenated selectively at the C = C double
bond to give the saturated alcohols. The C = C double bond of terpene
ketones and related ketones containing the a,{3-unsaturated double bond were
selectively hydrogenated to give saturated ketones, which were further reduced to secondary alcohols. Furthermore, dihydrocarveols and shisools containing the isopropenyl group were hydroxylated at the C-8 position to give
the corresponding 8-hydroxy compounds. [2- 2H]-I-Carvone was also easily
biotransformed by Euglena as well as l-carvone and gave [2}H]-8-hydroxy-dneodihydrocarveol. On the basis of IH-NMR spectral data of [2- 2H]-8hydroxy-d-neodihydrocarveol, we consider that the hydrogen addition to the

234

y. Noma and Y. Asakawa

Table 7. Microbiological reduction, oxidation, and hydrolysis pattern for terpenoids and related
compounds by Euglena gracilis Z. (Y. Noma, unpubl. data)

A. Microbiolgical reduction
1. Reduction of aldehydes to alcohols
a) Reduction of terpene aldehydes to terpene alcohols
Myrtenal (1) Myrtanal (5) l-Perillaldehyde (7a) dl-Perillaldehyde (7a and 7b)
trans- & cis-l,2-Dihydroperillaldehydes (14 and 15) l-Phellandral (18)
trans- & cis-Tetrahydroperillaldehydes (23 and 24) Cuminaldehyde (27)
Citral (30 and 31) d- and l-Citronellal (38 and 39) dl-Citronellal (38 and 39)
b) Reduction of aromatic and related aldehydes to alcohols
Benzaldehyde (46) 0-, m- & p-Hydroxybenzaldehydes (47-49)
0-, m- & p-Anisaldehydes (50-52)
0-, m- & p-Chlorobenzaldehydes (53-55)
2-,3-, & 4-Cyanobenzaldehydes (56-58) 0-, m- & p-Nitrobenzaldehydes (59-61)
0-, m- & p-Tolualdehydes (62-64)
o-Phthalaldehydic acid (65)
Terephthalaldehydic acid (66) o-Phthalaldehyde (67) Isophthalaldehyde (68)
Terephthalaldehyde (69) o-Vanillin (70) 3-Nitrosalicylaldehyde (71)
Vanillin (72) Veratraldehyde (73) Isovanillin (74) Ethylvanillin (75)
2-Hydroxy-5-methoxybenzalsehyde (76) 2,4-Dimethylbenzaldehyde (77)
2,3-,2,4-,2,5-, and 3,5-Dimethoxybenzaldehydes (81-84)
2,3,- 2,4-, 2,6-, 3,4-, and 3,5-Dichlorobenzaldehydes (85-89)
2,3,4-, and 3,4,5-Trimethoxybenzaldehydes (90 and 91)
p-Acetylamidebenzaldehyde (92) Phenyl acetaldehyde (93)
2-, and 3-Phenylpropionaldehydes (94 and 95) Cinnamic aldehyde (96)
a-Methylcinnamic aldehyde (97) Nicotin aldehyde (98)
c) Reduction of aliphatic aldehydes
n-Heptylaldehyde (99) Pelargonaldehyde (100) trans-2-Hexenal (101)
trans-2-Heptenal (102) cis-4-Heptenal (103) trans-2-Decenal (104)
2,4-Hexadienal (105) trans,trans-2,4-Heptadienal (106)
trans,trans-2,4-Nonadienal (107)
2. Reduction of ketones to alcohols
a) Reduction of saturated ketones
d-Dihydrocarvone (U4) I-Dihydrocarvone (124') d-Isodihydrocarvone (U5)
I-Isodihydrocarvone (U5') d-Carvomenthone (144) I-Carvomenthone (144')
I-Isocarvomenthone (146) d-Camphor (250) I-Camphor (250')
dl-Camphor (250 and 250') 2-,3-, and 4-Methylcyclohexanones
Cyclobutanone Cyclohexanone (157) Cycloheptanone
Cyclooctanone Cyclodecanone Cyclododecanone
d-Dihydrocarvone-8,9-epoxides (150a and 150b) d-Isodihydrocarvone )-8,9-epoxides
I-Dihydrocarvone-8,9-epoxides (150a' and 150b')
2-Butanone (229)
2-Pentanone (230) 2-Hexanone (231) 2-Heptanone (232)
2-0ctanone (233) 2-Nonanone (234) 2-Decanone (235)
2-Undecanone (236) 2-Dodecanone (237)
7,8-Dihydrobenzalacetone 7,8-Dihydrobenzalacetophenone
4-(p-Hydroxyphenyl)-2-butanone (222) I-Phenyl-3-hydroxyhexa-5-one (223)
Benzoylacetone (226) 4-0xodihydroisophorone
b) Reduction of a/i-unsaturated ketones
a-Ionone (166) j3-Ionone (167) Acetophenone (174)
0-, m- & p-Hydroxyacetophenones (176-178)
0-, m- & p-Methoxyacetophenones (179, 181, and 183)
p-Methylacetophenone (189) Acetovanillone (198)
2,5-Dimethylacetophenone (193) 3,4-Dimethoxyacetophenone (192)
Propiophenone (208) Butyrophenone (2U)
3. Hydrogenation of olefins
a) Hydrogenation of olefin conjugated with carbonyl group
I-Carvone (U3) d-Carvone (U3') I-Carvotanacetone (143)

Euglena gracilis Z

235

Table 7. Continued

B.

C.

D.

E.

d-Carvotanacetone (143') I-Carvone-8,9-epoxides (149a and 149b)


d-Carvone-8,9-epoxides (149a' and 149b') 2-Methyl-2-cyclohexenone (153)
2-Cyclohexenone (156) a-Ionone (166) f3-Ionone (167)
Benzalacetone (168) Benzalacetophenone (169)
2' -Hydroxychalcone (171) 3-Buten-2-one (173) 4-0xoisophorone
b) Hydrogenation of olefin not conjugated with carbonyl group
Cinnamyl alcohol (119) 2-Methylcinnamyl alcohol (121)
a-Ionone (166) f3- Ionone (167)
Microbiological oxidation
1. Oxidation of alcohols
a) Oxidation of primary alcohols to aldehydes and acids
b) Oxidation of secondary alcohols to ketones
I-trans-Carveol (134) d-cis-Carveol (135') d-Isoborneol (249')
Cyclobutanol Cyclopentanol Cyclooctanol Cyclodecanol
*Diastereo- and enantioselective dehydrogenation is observed in carveol, borneol and
isoborneol.
2. Oxidation of aldehydes to acids
Myrtenal (1) Myrtanal (5) I-Perillaldehyde (7a) dl-Perillaldehyde (7a and 7b)
frans- & cis-l,2,-Dihydroperillaldehydes (14 and 15) I-Phellandral (18)
frans- & cis-Tetrahydroperillaldehydes (23 and 24) Cuminaldehyde (27)
d- and I-Citronellal (38 and 39) dl-Citronellal (38 and 39) Benzaldehyde (46)
m- & p-Chlorobenzaldehydes (54-55) m- & p-Tolualdehydes (63-64)
2-, and 3-Phenylpropionaldehydes (94 and 95) Pelargonaldehyde (100)
*Acids were obtained as minor products.
3. Hydroxylation
a) Hydroxylation of nonallylic carbon
b) Hydroxylation of allylic carbon
d- and I-Limonenes (136' and 136) l-Methyl-l-cyclohexene Cyclohexene
4. Oxidation of olefins
a) Formation of epoxies and oxides
b) Formation of diols
c) Formation of trio Is
5. Lactonization
Hydrolysis
a) Hydrolysis of ester
d-trans- & cis-Carveyl acetates (138' and 139') I-cis-Carveyl acetate (139)
I-cis-Carveyl propionate (140) Geranyl acetate
Hydration
a) Hydration of C = C bond in isopropenyl group to tertiary alcohol
d-Neodihydrocarveol (126) I-Dihydrocarveol (127) d-Isodihydrocarveol (128)
I-Neoisodihydrocarveol (129) I-Neodihydrocarveol (126') d-Dihydrocarveol (127')
1- IsodihydrocarveoJ(128, ) I-Neoisodihydrocarveol (129')
frans- & cis-Shisools (9 and 10)
Isomerization
Geraniol (32) Nerol (33)

carbon-carbon double bond at the C 1 and C2 position by Euglena occurred


from the si face and re face, respectively, namely, anti addition. In the
hydroxylation process, l-aminobenzotriazole used as cytochrome P-4S0 inhibitor did not affect inhibition. In biotransformations of cycloalkanones and
cycloalkanols Euglena reduced cycloalkanones to the corresponding
cycloalkanols and the order of preference for the reduction was C4 > C7 > C6

236

y. Noma and Y. Asakawa

> C12 > C8 > ClO, whereas that for the dehydrogenation of the cycloalkanols
was C5 > C4 > C10 > C8 > C7 > C12. When both cyclopentanol and
cyclohexanone were added to the cultured broth at the same time, both
dehydrogenation and reduction were observed at the same time. For dl-aionone, and j3-ionone, Euglena preferred reduction of the carbonyl group to
hydrogenation of the C = C double bond. In the case of cinnamic aldehyde
and a-methylcinnamic aldehyde, the reduction of the aldehyde group occurred predominantly, and then the resulting alcohols were further hydrogenated to the corresponding saturated alcohols. However, in the case of
benzalacetone and benzalacetophenone, hydrogenation of the C = C double
bond in the side chain occurred predominantly rather than the reduction of the
carbonyl group. Acetophenone and related ketones were also reduced
nons electively to the corresponding secondary alcohols. Aliphatic methyl
ketones were also reduced. l-trans- and d-cis-Carveols and d-isoborneol were
enantio- and diastereoselectively transformed. Terpene esters such as carveyl
acetates and carveyl propionates were hydrolyzed. Terpene hydrocarbons and
related hydrocarbons such as limonene, l-methyl-l-cyclohexene, and
cyclohexene were also biotransformed to give the hydroxylated compounds.
Euglena is considered a good bioreactor to prepare primary alcohol and
secondary alcohol from aldehydes and ketones, respectively. Among the
metabolites, very important fragrant components such as shisool, etc. have
been obtained. Microbiological reduction and oxidation pattern for terpenoids
and related compounds by Euglena gracilis Z is summarized in Table 7.
Acknowledgements. The authors would like to thank Professor Emeritus Shozaburo Kitaoka and

Professor Nagahisa Nakano (University of Osaka Prefecture), Dr. Toshihiro Hashimoto and Dr.
Hironobu Takahashi (Tokushima Bunri University), and Professor Toshifumi Hirata (Hiroshima
University) for useful discussions, and Ms. Maoko Miki, Akiko Sogo, Sachiko Fujii, Yasuko
Wakita, and Hitomi Sakamoto for technical assistance.

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essential oils and aromatics of Japan, Utsunomiya, pp 367-368
Schiff JA, Lyman H, Russell GK (1971) [12] Isolation of mutants from Euglena gracilis. Methods
EnzymoI23:143-162
Sudate Y, Goto M (1986) Production of arachidoenic acid by Euglena. Proc 2nd Annu Meet
Euglena Res Assoc, Osaka, Abstr Pap, pp 17-18
Tani Y, Tsumura H (1989) Screeing for tocopherol-producing microorganisms and a-tocopherol
production by Euglena gracilis Z. Agric Bioi Chern 53:305-312
Tani Y, Okumura M, Li S (1987) Liquid wax ester production by Euglena gracilis. Agric Bioi
Chern 51:225-230
Yamada T, Maekawa F, Egami F, Yasugi R (eds) (1983) Dictionary of biology, 3rd edn. Iwanami
shoten (Tokyo)

XII Haplophyllum patavinum (L.) G. Don fil.


(Paduan rue): In Vitro Regeneration, and the
Production of Coumarin Compounds
E.M. CAPPELLETTI\ G.
and A. PIOVAN 2

INNOCENTI2 ,

R.

CANIAT0 1,

R.

FIUPPINI\

1 Introduction
1.1 Classification, Distribntion, and Importance of the Plant

The genus Haplophyllum belongs to the family Rutaceae. More than 70 species, growing from the Mediterranean region to eastern Siberia (most of them
in western and central Asia), are assigned to this genus (Everett 1981). Only
eight species can be found in Europe (Townsend 1968); H. patavinum (L.) G.
Don fil. is the only species occurring in Italy (Townsend 1968; Pignatti 1982).
H. patavinum was first collected on the Euganean Hills (NE Italy), probably in 1722, by the Italian botanist Micheli, who described it as Pseudo-Ruta
patavina (Micheli 1729) and stressed the differences from the genus Ruta
("plantae genus a Ruta diversum"). This species, described by Zanichelli
(1730) as Pseudoruta Micheli, was then included by Linnaeus (1753) in the
genus Ruta (Ruta patavina L.). De lussieu (1825) split the genus Ruta into
two genera, Ruta and Aplophyllum; the plant species from the Euganean Hills
was assigned to the latter genus by Don (1831). Spach (1849) changed
Aplophyllum into Haplophyllum.
At present, there is general agreement regarding Haplophyllum as a genus
distinct from Ruta, on the basis of morphological characters and chemical
evidence (Waterman 1975).
H. patavinum (Fig. 1) belongs to the section Oligoon of the genus
Haplophyllum (dehiscent fruit with five carpels and two ovules in each
loculus) according to Vvedensky (1949). It is a perennial herb with yellow
flowers in dense cymose inflorescences; leaves crowded, basal leaves simple,
middle leaves 3-sect to the base, uppermost leaves linear, simple, or 3-sect.
The leaf morphology and also the leaf width are very varied.
Two varieties of H. patavinum were described. The variety albanicum of
Baldacci (1901) later described as a separate species, H. albanicum (Bald.)
Bornm., is now regarded as a synonym of H. boissieranum Vis. & Pancic
(Townsend 1968). According to Beguinot (1905), the wide variability in leaf
width and the occurrence of many intermediate forms do not support the
variety angustifolia (Ruta patavina L. var. angustifolia Nob.).

1
2

Department of Biology, University of Padua, Via U. Bassi 58/B, 35131 Padua, Italy
Department of Pharmaceutical Sciences, University of Padua, Via Marzolo 5, 35123 Padua, Italy

Biotechnology in Agriculture and Forestry, Vol. 41


Medicinal and Aromatic Plants X (ed. by Y.P.S. Bajaj)
Springer-Verlag Berlin Heidelberg 1998

Haplophyllum patavinum (L.) G. Don til. (paduan rue)

239

Fig. 1. Haplophyllum patavinum (L.) G. Don til.

The chromosome number of H. patavinum is 2n = 18 (Darlington and


Wylie 1955).
H. patavinum has a discontinuous distribution with a wide illyrian range
extending from Albania to Slovenia and a punctiform relict disjointed range
on the Euganean Hills. Further information and details on distribution of the
species can be found in Dolcher (1957). The plant grows in open habitats on
well-drained calcareous soils (dry and sunny slopes, edges of the roads, uncultivated stony fields) (Fig. 2).
In the punctiform relict Euganean range, H. patavinum forms endangered and highly unstable populations, which are facing slow extinction
as the result of habitat modification by man's activities and the peculiar propagation features of the species (see Sect. 1.2, following). The species has
been included in the Red Book of Italian threatened species (Conti et al.
1992).
To the authors' knowledge, no use of H. patavinum in folk medicine is
reported and no phytochemical investigation had been previously undertaken.
Nevertheless, this species is worthy of investigation, since biologically active
natural substances of potential pharmaceutical interest are known to occur in
the genus Haplophyllum, namely coumarin compounds (Murray et al. 1982;

240

E.M. Cappelletti et al.

Fig. 2. Habitat of H. patavinum. Locality Sassonegro near Arqua Petrarca (Padua)

Murray 1991), quinoline and other types of alkaloids (Openshaw 1967;


Ulubelen 1984, Ulubelen et al. 1994a and references therein), and lignans
(Ulubelen et al. 1994b and references therein).
Another species, H. tuberculatum (Forssk.) A. Juss., is used in Iraqi
and Saudi Arabian folk medicine for a variety of ailments including malaria,
rheumatism, gynecological troubles, as an aphrodisiac, and as a cure for scorpion stings (AI-Yahya et al. 1991). Some of its chemical constituents have
antimicrobial and antimalarial activities (Khalid et al. 1986); moreover, plant
extracts may provide a potential alternative for insect control (Mohsen et al.
1989).
The small size of H. patavinum populations and the consequent
scarcity of plant biomass available in the natural habitat had hitherto
hindered systematic chemical investigations. Therefore, in vitro culture
of this species has been undertaken to obtain additional plant material
and, in the meantime, to set up non-conventional practices for plant
propagation.
1.2 Conventional Propagation

Natural propagation of H. patavinum is mainly vegetative, by shoots arising


from the rhizome. Therefore, agricultural practices such as deep plowing lead
to the disappearance of the plant (Cappelletti 1957). In the natural habitat, the
production of viable seeds is very scarce, since frequent embryo abortion at
different developmental stages was observed (Cappelletti 1929; Guzzo et al.
1991).

Haplophyllum patavinum (L.) G. Don fil. (paduan rue)

241

Slightly greater amounts of viable seeds are produced by specimens transplanted to different soil (unfortunately, the transplanted specimens do not
survive for long), which supports the hypothesis put forward by Cappelletti
(1929) of sterility of mycotic origin. In plants of H. patavinum cultivated in the
Botanical Garden of Paduva, vegetative propagation occurs frequently, but
only one seedling was observed.
1.3 Productiou of Coumariu Compounds

Coumarin compounds were identified in all the families belonging to the


Rutales. Furocoumarins, pyranocoumarins, and other types of prenylated and
isoprenylated coumarins are of common occurrence in the Rutaceae, while
they are more rarely found in the other families of the same order. Many
coumarin compounds have been reported to occur in the genus Haplophyllum
(Table 1).
All the detected coumarins are oxygenated at C-7. In addition to
the well-known coumarins such as umbelliferone, herniarin, osthol, and
scopoletin, the occurrence of several new mono-, di-, and trioxygenated
coumarins has been reported. A number of glycosylated and/or acetylated
derivatives of scopolin (scopoletin-,B-D-glucoside) have been reported
(Murray 1989). A few pyranocoumarins, such as xanthyletin and seselin, have
been also isolated from Haplophyllum. Only in recent years have both linear
and angular furocoumarin derivatives been isolated.
No chemical investigation had been carried out previously on H.
patavinum. From the plants growing on the Euganean Hills we isolated only
the two hydroxycoumarins, umbelliferone (Innocenti et ai. 1993) and
scopoletin (Eilippini et aI., submitted).

2 In Vitro Approaches
2.1 In Vitro Culture Studies

To our knowledge, no reports on in vitro culture of Haplophyllum


species were available in the literature before our investigations on H.
patavinum; the reason for this may be that the biogenetic potentialities
and pharmaceutical applications of the genus Haplophyllum had been
underestimated.
The previous investigations by our research group on H. patavinum dealt
with in vitro seed germination and plant regeneration (Filippini et ai. 1992,
1994) and coumarin compounds production by callus cultures (Filippini et ai.
1993; Innocenti et ai. 1993).

Angustifolin
Herniarin

Obtusifolin

Tenuidin

Ramosinin

Ramosin

HC~

H?CY

OCH3

OH

OCH!

OH

OCH3

o~

II
0

c~

V~
V~

V~

H. glaberrimum

H. thesioides

H. obtusifolium

H. tenue

H. ramosissimum

H. ramosissimum

Gashimov et al. (1979b)

H. versicolor

Versicolin

Ulubelen et al. (1993c)


R6zsa et al. (1986)

Bessonova et al. (1988)

Abyshev et al. (1980)

Gashimov et al. (1979a)

Gashimov et al. (1979a)

Gashimov and
Orazmukhamedova (1978)

Gonzalez et al. (1973)


Innocenti et al. (1993)

Reference

Gonzalez et al. (1973)

Species

H. hispanicum

R,

Auraptene

R4

H. bungei

R3

Osthol

R2
H. hispanicum
H. patavinum

RJ

Umbelliferone

Monoxygenated

R1

Table 1. Distribution of coumarin compounds in Haplophyllum species

g.

'"0
'"0

0>

(')

tr1

tv

t!3

Pediccllone

6-Methoxymarmin
acetonide

6-Methoxymarmin

Geranylscopoletin

6-Methoxy-7-dimethyl
allyloxycoumarin

5,7Dihydroxycoumarin
[soscopoletin

5-Hydroxy-7methoxycoumarin
Scopoletin

Dioxygcnated

Table 1. Continued

R,

O~H

O~

0+

O~OH

OCH,

OCH

OCH,

OCH,

O~
OH

OCH

o~

OH

OCH.,

OH

R,

OH

OCH,

R,

OH

OH

OCH,

R,
Rj

cappadocicum
dauricum
hi'panicum
putavinum
pedicel/atum
ramosissimum
dauricum

H. pedicel/a tum

H. pedicel/atum

Kuznetsova and Gashimov (J 973)

Kagramanov et aJ. (1979)

Kuznetsova and Gashimov (1972)

Kuznetsova and Gashimov (1972)


H. pedicel/alum

Gonzalez ct aJ. (1973)


ll. pedicel/atum

Gashimov and
Orazmukhamedova (1978)
Matkarimov et al.
Gashimov ct al. (1979a)

Abyshev and Gashimov (1982)

Gashimov and
Orazmukhamedova (1978)
G(\zler et al. (1992)
Batirov et aJ. (1984)
Gonzalez et al. (1973)
Filippini et aJ. (submitted)
Kagramanov et aJ. (1979)
Bessonova et al. (1989)
Batsuren et aJ. (1982)

Reference

ll. hispanicum

H. obtusifo/ium
1-1. ramosissimum

ll. bunRei

H. bungei

H.
1I.
H.
II.
H.
H.
H.

II. bunRei

Species

P-

VJ

...

...,
c

:>

OJ

.::0
OJ

:!:

:>

CJ

;'!

;:::

S
'"

!:i'

'"

;'!

~
:::::
;:::

~c

OH

OH

OH

R.

R,

H. tenue

H. villosum
H. mirtifolium

H. ramosissimum

H. bungei

Vdovin et al. (1983)

Abyshev and Gashimov (1979d)

Abyshev et al. (1980)

Abyshev et al. (1980)

Bessonova et al. (1989)

Abyshev and Gashimov (1982)

Matkarimov et al. (1980c)

Bessonova et al. (1990)

H. ramosissimum
H. ramosissimum

Matkarimov et al. (1980a)

Matkarimov et al. (1980b)

Tikhomirova et al. (1977)

Reference

H. obtusifolium

H. obtusifolium

H. albert-regelli

Species

H. dauricum

C-~-D-g1ucosyl

OCH3

OCH3

OCH3

OCH3

OCH3

R3

Dauroside D

OCH3

OCH,

OCH,

O~H

OH

Jyc-o.gIUC05Y1

OH

JyOH

OH

O~OH

O~

R,

H. pedicellatum

H'~

OH

~~

aCH3

RJ

6-Geranyloxy-7 methoxycoumarin

Tenudiol

Villosin

Scoparon

Bungeidiol

Obtusoside

Obtusinin

Obtusinol

Collinin

Table L Continued

a.

(t

't:l
't:l

QO

(l

tTl

OH

OH

Obtusiprenol

Obtusidin

OH

OH

Capensin

Obtusiprenin

OH

OH

Obtusitsin

Fraxetin-7 -O-B-Dl-glucopyranoside

OH

OH

R,

Haptusinol

Obtusin

Obtusifol

5.6-Dimethoxy
auraptene

3-Methoxy-R-hydroxy
umbelliferone

Trioxygenated

Table 1. Continued

OCHo

OCH,

o~

OH

OCH

OCH,

OCH,

OCH

OCH

OCH,

OCH

OCH,

R,

O-J1.-D-glucosyl

OH

OH

o~

OH

O~

OH

o~

OH

R,

OH

H'~

HL

OCH,

R,

\/
c---.,:9

OCH,

R,

11. obtusifolium

11. obtusifolium

H. obtusifolium

H. obtusifolium

H. obtusifolium

H. obtusij()lium

H. obtusij()lium

H. obtusifolium

H. obtusifolium

H. hispanicum

H. schelkovnikovii

Species

Matkarimov et a!. (1982)

Batirov et aL (1982)

Batirov et a!. (1982)

Matkarimov et aL (1981)

Matkarimov et a!. (1981)

Batirov ct a!. (1980)

Abyshev and Gashimov (1979c)

Abyshev and Gashimov (I 979b )

Abyshev and Gashimov (1979a)

Gonzalez et a!. (1973)

Abyshev et a!. (1978)

Reference

.(:>.
U,

Q..

'C"
::>
'"
...,

'0

::>

:E

S
'"
:::
2l

'"

""'"

:::

:::::
2l

'<

.g;::-

::t:
-

Thesiolen

Furocoumarins

Marmesin

Ptilin

Ptilastin

Ptilastol

Dihydrofurocoumarins

H3C _

C9

CH ,

,..

~' I

OCH

sO

OR

C~'--<=C(\
I

H2C-Y

HO-c

R=

R=

R=

;:-.... I

eM,

C-l,

H3

Sese lin

000

Xanthyletin

Pyranocoumarins

Table 1. Continued

If. thesioides

If. glaberrimum

H. ptilostylum

If. ptilostylum

If. ptilostylum

H. dubium
If. dshungaricum
If. multicaule
If. per!oratum
If. thesioides

If. cappadocicum

If. dshungaricum
If. multicaule

Species

Ulubelen et al. (1993c)

Rozsa et al. (1986)

Ulubelen et al. (1993b)

Ulubelen et al. (1993a)

Ulubelen et al. (1993a)

Gazier et al. (1992)


Tikhomirova et al. (1977)
Timomirova et al. (1974)
Timomirova et al. (1974)
Razakova and Bessonova (1989)
Ulubelen et al. (1993c)

Timomirova et al. (1974)


Timomirova et al. (1974)

Reference

"""

?'-

2.

CD

-0
-0
~

'"

!Tl

Haplophyllum patavinum (L.) G. Don fil. (paduan rue)

247

2.2 Establishment of Tissue Cultures and Cell Suspensions

2.2.1 Callus Culture

Native plants of H. patavinum from the Euganean Hills and sterile


plantlets grown from seeds produced by plants cultivated in the Botanical
Garden of the University of Padua were used as starting materials. The plants
were sterilized by treatment with ethanol (70%) for 20s and Ca(OCI)2 7%
(w/v) for 10min, then washed 3x with sterile distilled water. Primary
explants from leaves were introduced into culture on BS medium (Gamborg
et al. 1968) containing sucrose (30 gil) and agar (8 gil), supplemented with
2,4-D (2mg/l), NAA (O.Smg/l), IAA (O.Smg/l), and kin (0.2mg/l), according
to Fischer et al. (1988). The pH of the medium was adjusted to S.7. Calli were
also induced under the same conditions from in vitro-raised seedlings (see
Sect. 2.3.1). The induced callus tissues were grown in the dark at 2S C and
subcultured on fresh media every 4 weeks. Calli were obtained with
high frequency (about 9S% of explants) from both explant types. Evident
differences were immediately revealed for callus characteristics: calli obtained
from native plants showed yellow color, good friability, and high growth
rate; in contrast, calli obtained from sterile seedlings showed white-yellow
color, hard consistence, and slow growth rate (Fig. 3). After five subcultures,
on account of their unsuitable characteristics, the tissue cultures obtained
from sterile seedlings were stopped. After about 20 subcultures, the calli
obtained from native plants showed two different cell strains: calli with
white-yellow color, good friability, and very high growth rate (strain A),
and calli with dark yellow color, hard consistence, and good growth rate
(strain B).
2.2.2 Suspension Cultures

Cell suspension cultures were initiated from well-established strain A


and strain B calli. Suspension cultures were grown in SOO-ml Erlenmeyer
flasks containing 1S0ml liquid BS basal medium supplemented with
sucrose (30g/l) and 2mg/l 2,4-D, O.Smg/l kin, 0.5mg/l NAA, and 0.2mg/l
IAA at pH 5.7. The growth curves of suspension cultures were calculated
on the basis of fresh weight (FW), dry weight (DW) and cell volume
after sedimentation (CVS) (Fig. 4). Homogeneous suspensions were
obtained only from strain A calli; the dark, hard calli (strain B) failed
to produce suspensions. Light conditions are known to stimulate coumarin biosynthesis, at least in some species (Dhillon and Brown 1976);
cell suspensions were kept in the dark and also under a 12-h
photoperiod.

248

E.M. Cappelletti et a!.

2.3 In Vitro Propagation

2.3.1 Propagation from Seeds Germinated in Vitro


In some species, germination of seeds is known to be greatly increased by the
use of in vitro methods (Fay 1992). Previous research (Filippini et al. 1994)
showed that germination of seeds from plants of H. patavinum grown in the
Botanical Garden of Padua was greatly increased by in vitro culture. After
scarification in conc. H 2S04 and sterilization in 7% (w/v) Ca(OCI)z, the seeds
were kept on B5 solid medium containing 3% sucrose and solidified with 0.3%

Fig. 3A,B. H. patavinum. A Calli 2 years old obtained from native plants. B Calli 4 months
old obtained from in vitro-grown seedling, grown on BS medium supplemented with 2,4-D
(2mg/I), NAA (O.Smg/I), IAA (0.5 mg/l) , kin (0.2mg/I); 3% sucrose. (E.M. Cappelletti et a!.,
unpub!.)

Haplophyl/um patavinum (L.) G. Don fil. (paduan rue)

249

10

15

20

25

30

35

40

days
Fig. 4. The growth curves of 4-month suspension cultures subcultured in B5 medium supplemented with 2,4-D (2 mg/I), NAA (0.5 mg/I), IAA (0.5 mg/I), kin (0.2 mg/I); 3 % sucrose calculated
after cell fresh weight (-D-), cell dry weight (-0-), and cell volume after sedimentation (-6-).
(E.M. Cappelletti et aI., unpubl.)

(w/v) Gelrite for developing the plants. After 2 weeks, every seed was
wetted with 20,u1 of kinetin (1 mg/ml). All seeds germinated within 4 weeks.
Therefore, the in vitro germination of seeds from plants growing in
the natural habitat of the Euganean Hills was also investigated. The very
scarce seed production was the main obstacle to this in vitro germination
research. In fact, the small amount of available seeds severely limited the
number of culture media tested and prevented the statistical evaluation of
the results. The effect of scarification on seed germination was first investigated. To evaluate the effect of scarification (cone. H 2S04 treatment) on
seed germination, scarified and nonscarified seeds were sown on B5 medium
without sucrose and hormones. Scarification proved to be a basic requirement for successful seed germination; in fact the nonscarified seeds failed to
germinate.
The effects of sucrose (3%) addition to the B5 medium, and kinetin and
gibberellic acid (20,u1 of a I-mg/ml solution) treatments were also investigated
using scarified seeds. The results are quoted in Table 2. Germination was not
influenced by sucrose or hormone addition to the medium.

250

E.M. Cappelletti et al.

Table 2. Effects of sucrose, kinetin, and gibberellic acid (GA3) on

seed germination

Sucrose

Kinetin

+
+
+

GA3

Seed
germination( % )
65

50
60
55
50
50

Contamination by bacteria and fungi was often observed in cultures


following seed coat break and seedling emersion. Additional sterilization of
the plant material was therefore needed to avoid contaminants overwhelming
the plant culture. Contamination was never observed in dishes containing
nongerminating (not scarified) seeds, which suggests that contaminants do not
derive from inadequate external disinfestation of seeds, but from endogenous
(endophytic) microorganisms.
2.3.2 Micropropagation from Nodal Shoot Segments

Regeneration from nodal shoot segments has been described (Filippini et al.
1994). The source of nodal shoot explants was in vitro-raised seedlings, 10-12
weeks old. The most abundant axillary shoot proliferation was induced by
cultivating the explants (0.5-1.5 cm long) on MS (Murashige and Skoog 1962)
with 3% sucrose, 0.8% agar, 1mg/12,4-D, and 0.2mg/1 kin, pH 5.7 at 25C in
a 12-h photoperiod. A much lower incidence of shoot formation was observed
when 0.1 mg/l kinetin was added and when B5 medium supplemented with the
same hormone amounts was used. Shoots were subcultured into fresh medium
after 6 weeks for further proliferation of axillary shoots. Rooting was achieved
by transferring the isolated shoots on MS supplemented with 3% sucrose,
0.8% agar, 0.5mg/1 BAP, and 1mg/1 IAA at 25C in the dark for 12h and then
under a 12-h photoperiod. After 2 weeks they were transferred to halfstrength MS. About 70% of the isolated shoots rooted on this medium (Fig. 5);
the percentage root induction was low on the other media. Hardening of plants
before transplantation was found to be essential (Filippini et al. 1994).
2.3.3 Micropropagation through Callus Organogenesis

Micropropagation through organogenesis from callus has also been investigated; this procedure involves three stages, namely culture initiation, adventitious shoot formation, and rooting of the adventitious shoots.
Strain A and strain B calli were used for inducing adventitious shoot
formation on different media and under light condition (12-h photoperiod).

Haplophyllum patavinum (L.) G. Don fil. (paduan rue)

251

Fig. SA,B. In vitro regeneration of H. pata vinum from nodal shoot segments. A Axillary shoot
development. B Rooting of the axillary shoots. (Filippini et al. 1994)

Shoot formation was obtained only from the dark yellow hard calli (strain B)
(Table 3). The best results (low induction time, high shoot number, high
growth rate) were obtained on MS medium supplemented with IAA (3mg/l)
and kin (1 mg/I).
When the stems of the adventitious shoots had reached a length of about
5 mm, they were more or less dissected from the callus and transferred individually to a series of substrates with different hormone combinations for
shoot rooting (Table 4). For each substrate, two series of tests were carried
out: one directly exposed to a 12-h photoperiod, the other kept in the dark for
24h before light exposure (12-h photoperiod) to prevent possible auxin degradation. In any case, no root formation was observed, but only shoot proliferation and callus growth.
2.3.4 Somatic Embryogenesis

Both callus types (strains A and B) were cultivated on either solid or liquid MS
media with different growth regulator contents under a 12-h photoperiod at
25C to promote somatic embryogenesis (Table 5). The suspension cultures

252

E.M. Cappelletti et al.

Table 3. Growth media, auxin/cytokinin combinations and their effect on adventitious shoot
formation in two cell lines of H. patavinum

Cell line

Medium

Auxin
(mg/I)

Kinetin
(mg/I)

Strain A

B5

2,4-D (0.5)
NAA (0.5)
IAA (3)
2,4-D (0.5)
NAA (0.5)
IAA (3)

(0.5)

B5
MS
MS
MS
'MS 1/5 Ca'+
Strain B

B5
B5
MS
MS
MS
"MS 1/5 Ca2+

2,4-D (0.5)
NAA (0.5)
IAA (3)
2,4-D (0.5)
NAA (0.5)
IAA (3)

Shoot
induction

(1)
(0.5)
(1)

(0.5)
(1)
(0.5)

(1 )

++
+
+

, MS medium with 1/5 of CaCI, concentration (66.44mg/l).


- = shoots were not developed.
+ = relatively good shoot induction.
+ + = optimum shoot induction (high shoot number, low induction time, high growth rate).

were kept on a rotary shaker (80rpm). After 2 months, calli and suspension
cultures were transferred to solid or liquid hormone-free media, respectively,
to stimulate embryo development. Calli were also transferred to liquid media
and suspensions cultured to solid media. All these experiments failed to induce somatic embryogenesis.
Fairly high-frequency embryoids were produced in suspension cultures of
calli originated from in vitro-induced shoots as starting material. The abovementioned calli were obtained on hormone-free solid MS medium in the dark.
Suspension cultures were initiated, after three callus subcultures, in liquid
hormone-free MS medium under a 12-h photoperiod. Somatic embryos were
heart-shaped (Fig. 6).
Somatic embryogenesis was obtained in complete absence of exogenous
plant growth regulators; an analogous result was previously reported for another plant belonging to the same family, Citrus sinensis (L.) Osbeck (Vardi et
al. 1975).
2.4 Extraction and Structure of Coumarin Compounds

2.4.1 Extraction

Extractions of calli and suspension culture cells were carried out taking into
account the fact that coumarin compounds may be present in both free and

Haplophyllum patavinum (L.) G. Don fil. (paduan rue)

253

Table 4. Auxin/cytokinin/gibberellin combination of different growth media

Medium

Auxin
(mg/I)

Cytokinin
(mg/I)

MS (P)
MS (P)
MS (P)
MS (P)
MS (P)
MS (P)
MS (P)
MS (P)
MS (P)
MS (P)
MS (P)
B5 (dilution 1: 10) (P)
B5 (dilution 1: 10) (P)
B5 (dilution 1: 10) (P)
B5 (dilution 1: 10) (P)
Water (A)
Water (A)
Water (A)
Water (A)
MS (dilution 1: 2) (P)
MS (dilution 1:2) (P)
MS (dilution 1: 2) (P)
MS (dilution 1: 2) (P)
Water (A)
Water (A)
Water (A)
Water (A)

IAA (1)
IBA (0.2)
IBA (1)
IBA (3)
IBA (5)
IBA (0.2)
IBA (1)
IBA (3)
IBA (5)
IAA"
IBN
lBA (1.5)
IBN
IBA (1.5)
IBN
IBA (1.5)
IBA"
IBA (1.5)
IBA"
lBA (1)
IAA (1)
IBA (1)
IAA (1)
lBA (1)
IAA (1)
lBA (1)
IAA (1)

BAP (0.5)

BAP
BAP
BAP
BAP

Gibberellin
(mg/I)

Sucrose
(mg/I)

GA,
GA,
GA,
GA,
GA,
GA,
GA,
GA,

30
30
30
30
30
30
30
30
30
30
30
30
30
0
0
30
30
0
0
30
30
30
30
30
30
30
30

(0.5)
(0.5)
(0.5)
(0.5)

BAP (0.5)
BAP (0.5)
BAP (0.5)
BAP (0.5)

(1)
(1)
(1)
(1)
(1)

(1)
(1)
(1)

, Shoots excised were treated directly with IAA or IBA powder (Sigma). (P)
(A) = Agar.
Table 5. Auxin/cytokinin
embryogenesis

combinations

tested

Phytagel.

to

induce

Auxin
(mg/I)

Cytokinin
(mg/I)

2,4-D (5)
NAA (0.5)
2,4-D (2) + NAA (2)
2,4-D (1)
2,4-D (0, 11)
IAA (1)
IAA (0.5)
2,4-D (1)
IAA (3)

Kin (0.5)
BAP (0.5)
Kin (2)
Kin (1)
Kin (1)

Kin (1)

bound forms (Murray et al. 1982). Preliminary research on in vitro


cultures (tissue and cell cultures) of H. patavinum, however, showed that
coumarin derivatives occur only in the free form; therefore, the extraction
procedure involving acid hydrolysis, previously suggested for optimum

254

E.M. Cappelletti et al.

Fig. 6. Heart-shaped somatic embryo of H. patavinum. (E.M. Cappelletti et aI., unpubl.)

coumarin compound recovery from Coronilla (Innocenti et al. 1996; Piovan et


al. 1996), was not followed.
Calli. The fresh callus tissues (strain A and strain B) were homogenized and
extracted with methanol in a Soxhlet extraction apparatus for 48 h; the
methanolic extract was then concentrated under vacuum. The methanolic
solution was analyzed.
Solid Media. In Haplophyllum tissue cultures the characteristic coumarin nucleus fluorescence in the medium, a result of natural excretion of secondary
products from the cells, was observed (Filippini et al. 1993; Innocenti et al.
1993). The media were refluxed for 1 h in methanol. The aqueous-methanol
solution, after filtration, was concentrated to 50-100ml and, following a modified Spath method (1937), supplemented with aqueous KOH (10%) and extracted with ethyl ether at room temperature; then the aqueous phase was
acidified with Hel. The acidic solution was extracted with ethyl ether (48h).
After distillation of the solvent under vacuum, the residue was dissolved in
methanol and analyzed (Filippini et al. 1992).
Suspension Cultures. After centrifugation at 5000rpm for 20 min, the
supernatant (medium) was extracted with ethyl ether (48h). After distillation
of the solvent under vacuum, the residue was dissolved in methanol and
analyzed. The cells were frozen-dried and treated following the above described procedure for calli.

Haplophyllum patavinum (L.) G. Don til. (paduan rue)

Ho~oAo

255

---.
o

Umbelliferone (I)

Osthenol (II)

Angelicin (IV)

Columbianetin (III)

Fig. 7. Biosynthetic pathway to angelicin

2.4.2 Analysis and Structure

The methanolic solutions were analyzed by TLC on analytical silica gel plates
(Merck, Cat. no. 5715) using CHCl3 or EtOAc-cyclohexane in different ratios
(3:1,2:1,1:1; v/v) as eluents.
Several coumarin compounds were isolated; five of them were identified
by their UV and MS spectra, and by comparison with authentic samples
(supplied by the Department of Pharmaceutical Sciences, University of
Padua). The purity of the compounds was controlled by HPLC under the
following conditions: column LiChrosorb RP 8 7 f1 (Merck), mobile phase
AcCN:H 20 (25:75; v/v); flow rate 1.5mllmin.
Two furocoumarins, columbianetin and angelicin (Fig. 7, III and IV), and
three simple coumarins, umbelliferone (7-hydroxycoumarin), osthenol (7hydroxy-8-prenylcoumarin) (Fig. 7, I and II) and 7-prenyloxycoumarin were
identified.
Umbelliferone: UV max (EtOH) 248, 258, 327nm; MS 70eV at M+ (m/z
162); prominent fragment ions at mlz 134 [M-COt, 105 [M-CHOt, 106 [MCOlo (relative abundance 55, 95, 70, and 60%).
7-prenyloxycoumarin: UV max (EtOH) 203, 249, 259, 322nm; MS 70eV at
M+ (mlz 230): prominent ions at 215 [M-CH3t, 213, 202 [M-COr, 187, 175,
162 [M-CsHs accompanied by hydrogen migrationt (relative abundance 88,
62, 50, 43, 80, and 90%).

256

E.M. Cappelletti et al.

Osthenol: UV max (EtOH) 203, 248, 258, 324nm; MS 70eV at M+ (mlz


230): prominent fragment ions at 213 [M-OHr, 187, 175 (relative abundance
60, 60, 46, and 100%).
Columbianetin: UV max (EtOH) 208, 252, 262, 327nm; MS 70eV at
M+ (m/z 246): prominent fragment ions at mlz 213 [M-HzO-CH3r, 188
[M-C3H60r, 187 [M-C3H70r, 160, 131 (relative abundance 34, 22, 60, 87, 27,
and 16%).
Angelicin: UV max (EtOH) 205, 243, 247, 299nm; MS 70eV at M+ (m/z
186): prominent fragment ions at 158 [M-COr, 130 [M-2CO and/or with H
rearr.r, 118 [M-C30 Z with 2H rearr.r, 102 (relative abundance 100,65,58,10,
and 45%).
Tissue Cultures. The coumarin compounds recovered from calli and media of
the two investigated cell strains are shown in Table 6.
The white-yellow friable calli (strain A) are characterized by poor biogenetic potentialities, at least as far as the coumarin compounds are concerned; in fact, only umbelliferone was detected in both callus and medium.
The dark yellow hard calli (strain B) accumulate umbelliferone and
its prenyl derivative 7-prenyloxycoumarin, whilst in the medium all the
compounds of the biosynthetic pathway of the angular furocoumarins,
namely umbelliferone, osthenol, columbianetin, and angelicin (Fig. 7) were
detected. This biosynthetic pattern in vitro is rather unexpected, since in
the starting plant material only umbelliferone was present (Innocenti et al.
1993).
Cell Suspensions. The biosynthetic potential of H. patavinum suspension cultures is quite similar to that exhibited by the same cell strain on solid medium:
only umbelliferone was recovered from cells and medium; the coumarin
biosynthetic capability does not seem to be influenced by light exposure (Table 6). Unfortunately, the cell line with strong coumarin biosynthesis (strain B)
is quite unsuitable for suspension cultures.
Isolation and chemical characterization of other classes of metabolites
biosynthesized by suspension cultures suggest that H. patavinum cells of strain
A produce also lignanolide compounds.

Table 6. Coumarin compounds produced by H. patavinum in vitro


Tissue cultures
Strain B

Strain A

Strain A

Dark

Dark

Dark

Light

Callus
Umbelliferone
Osthenol
7-Prenyloxycoumarin
Columbianetin
Angelicin

Suspension cultures

Strain A

Medium

Callus

Medium

+
+
+
+

Cells Medium Cells Medium

Haplophyllum patavinum (L.) G. Don fil. (paduan rue)

257

3 Conclusion
In vitro culture of H. patavinum has to be considered as a useful tool for the
conservation of this rare and endangered plant. Seed germination, which
occurs very rarely under natural conditions, is considerably enhanced in vitro,
allowing successful establishment of plants. This fact is particularly important
when the very low viable seed production from the native populations in the
Euganean Hills is considered. Moreover, seeds are preferred to vegetative
material as the source of propagation material because a wider genetic base
can be maintained (Fay 1992).
H. patavinum can be multiplied in vitro from nodal shoot segments, a
protocol which involves regeneration from existing meristems.
In comparison with other Haplophyllum species, H. patavinum is not
rich in coumarin compounds in vivo. However, a selected cell strain exhibited in vitro coumarin biogenetic potentialities stronger than in vivo.
In fact, new structures, not yet isolated in intact plants, are produced by
calli and excreted into the solid medium, from which all the intermediate
products of the biogenetic pathway of the angular furocoumarin, have been
isolated.
Although other angular furocoumarins have been detected from species
of the genus Haplophyllum (Table 1), the occurrence of the angular
furocoumarins columbianetin and angelicin, and the prenylated coumarin
osthenol had not previously reported.
Investigations on the biosynthesis of other classes of metabolites could
lead to the detection of other pharmaceutically active compounds, since preliminary unpublished data have shown the production of lignanolide compounds by H. patavinum in vitro.

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XIII Morus Species (Mulberry): In Vitro Culture,


Micropropagation, and the Formation of
Mulberrofuran, Kuwanol, and Other Secondary
Metabolites
y'P.S. BAJAJ\ J. IVANICKA 2 , and S. UEDA'

1 General Account
1.1 Botany, Distribution, and Importance of Morus Species

The genus Morus (family Moraceae) covers the numerous mulberry species
grown and cultured in various continents, mainly in Asia and Europe.
Koidzumi (1923) described 37 species, of which some common ones are given
in Table 1. The typical representative of diploid species (2n=28) is Morus
alba (white mulberry), which has long been cultivated in China, Korea, and
Japan for feeding silkworm (Bombyx mori L.). Cultivation of mulberry trees
is especially popular in the Jiangsu, Zhejiang, Fujian, and Sichuan districts
in China (Hotta 1951; Hotta et al. 1989). Another useful species, Morus
nigra (black mulberry), is a polyploid with 308 or 330 chromosomes (Agaev
1984).
Mulberries are anemophilous trees or bushes of vigorous branches and
spread crowns (Fig. 1). The trunk of the tree reaches 10m. The dentate leaves
are deciduous, light to dark green in color. Their inflorescences are represented by the male (Fig. 2A) and female (Fig. 2B) catkins which are
monoecious or dioecious. The female flowers form a thick multiple fruit (Fig.
2e). The actual fruits, little achenes or nutlets, are surrounded by the fleshy
sepals and grouped together with the fleshy axis to form the so-called syncarp
(Hill 1937). The white color of the unripe fruit changes to pink, red, or dark
red.
Mulberries are pomologically interesting for their edible sweet fruit.
In silviculture, although artificial diets for silkworms have been improved
(Ito et al. 1974, 1975), mulberry leaves are still essential. In Chinese
medicine, young twigs are applied for rheumatism and neuralgia. The leaves
are antifebrile. The fruits are nutritive and used in tonics. The root bark
is an antiinflammatory diuretic, a cough medicine, and a blood pressurelowering agent (Hotta et al. 1989). Examination of the biological activity
of Diels-Alder type morin, a light yellowish pigment in mulberry wood,

A-137 New Friends Colony, New Delhi 110065, India


Centre of Development of Horticulture, Hornonitrianska 20, 971-01 Prievidza, Slovakia
, Formerly: Faculty of Pharmaceutical Sciences, Kyoto University, Sakyo-ku, Kyoto 606-01, Japan
1

Biotechnology in Agriculture and Forestry, Vol. 41


Medicinal and Aromatic Plants X (ed. by y.P.S. Bajaj)
Springer-Verlag Berlin Heidelberg 1998

262

YP.S. Bajaj et al.

Table 1. Some important botanical species of mulberries


Species

Origin

Somatic
chromosomes
(2n)

Reference

Morus acidosa Griff.


M. alba L.
M. cathayane H.
M. indica L.
M. kagayame Koidz.
M. bombycis Koidz.
M. nigra L.

Australia
Central China
Japan
India
Japan
Japan, Korea
Persia

28
28
54,84, 112
28
28
28
308,330

M. tiliaefolia M.
M. rubra L.

Japan
North America

84
28

Katsumata (1979)
Bolkhovskikh et al. (1969)
Bolkhovskikh et al. (1969)
Bolkhovskikh et al. (1969)
Koidzumi (1923)
Bolkhovskikh et al. (1969)
Darlington and Wylie (1961);
Agaev (1984)
Bolkhovskikh et al. (1969)
Darlington and Wylie (1961)

Fig. 1. A MOTUS alba tree 20 years after differentiation from callus tissue induced from seedlings
(Kyoto University)

was found to inhibit lipid peroxidation in micelles (Wang and Zheng


1992), and in rat liver microsomes (Cholbi et al. 1991). Most recently, it
was found that morin hydrate exhibits myocardial salvage effects (Wu et al.
1995).

Fig. 2A-C. Reproductive organs of mulberries. A Staminate flowers. B Pistillate flowers. C Ripening fruits

264

Y.P.S. Bajaj et al.

1.2 Secondary Metabolites

Morus species, in addition to many other secondary metabolites, are a rich


source of intermolecular Diels-Alder-type adducts of prenylchalcone and
prenylated 2-arylbenzofuran. Morin (2' ,3,4' ,5,7-pentahydroxyflavone) was
first isolated by Perkin in 1895 (Harborne 1994). It was also obtained along
with maclurin (2,3' ,4,4' ,6-pentahydroxybenzophenone) as a constituent of the
heartwood of M. alba (Haley and Bassin 1951; Spada et al. 1956). From an
aqueous methanolic extract of mulberry leaves, quercetin, rutin, and
quercetin-3-triglucoside (moracetin) were obtained (Naito 1968a,b). Later,
four prenylated flavonoids, mulberrin, mulberrochromene, cyclomulberrin,
and cyclomulberrochromene, were isolated along with betulinic acid from the
stem and root bark of M. alba (Deshpande et al. 1968). Recent examination of
mulberry constituents revealed intermolecular Diels-Alder-type ad ducts comprising two molecules of prenylphenols (Nomura 1988). From the Japanese
Morus root bark, optically active kuwanon G was first isolatied as an active
substance exhibiting hypotensive action. On the basis of NMR data, this
compound was assumed to be a biogenetic intermolecular Diels-Alder type
adduct formed from dehydrokuwanon C and a chalcone derivative (Nomura
and Fukai 1980). From the leaves of M. alba infected with Fusarium solani f.
sp. mori, a natural Diels-Alder-type adduct chalcomoracin (CAL) was isolated
as a phytoalexin (Takasugi et al. 1980). About 40 kinds of optically active
Diels-Alder-type adducts have also been isolated from Japanese Morus root
bark and the Chinese crude drug Sang-Bai-Pi (Nomura 1988). The mulberry
Diels-Alder-type adducts may be classified into the following four groups on
the basis of their skeleton structures: (1) ad ducts of a chalcone and a dehydroprenylflavonoid (kuwanon G, kuwanon H, and sanggenon C), (2) ad ducts of a
chalcone and a dehydroprenylchalcone (kuwanon I and kuwanon J (KUJ), (3)
ad ducts of a chalcone and a dehydroprenyl-2-arylbenzofuran (CAL and
mulberrofuran C), and (4) ad ducts of a chalcone and a dehydro-prenylstilbene
(kuwanons X and Y) (Fig. 3). From the root bark of M. alba, j3-tocopherol was
obtained (Shibata et al. 1974). 1-Deoxy-nojirimycin was found in the leaves
and roots of Morus spp. (Yagi et al. 1976; Evans et al. 1985). Recently, Ncontaining sugars such as 1,4-dideoxy-1,4-imino-D-ribitol and their glycosides
as well as nor-tropane alkaloids such as calystegin B2 (1a,2/3,3a,4/3,tetrahydroxy-nor-tropane) have been isolated along with 1-deoxynojirimycin
from the root bark of M. bombycis (Asano et al. 1994a) and M. alba (Asano et
al. 1994b).

2 In Vitro Culture Studies


Extensive studies have been conducted on various species of Morus
on the establishment of callus culture, regeneration of plants, micropropagation, cryopreservation of germplasm, synthetic seed, and the forma-

265

Morus Species (Mulberry)

kuwanon G

kuwanon J

chalcomoracin

kuwanon X

Fig. 3. Typical mulberry Diels-Alder-type adducts

tion of secondary metabolites in cell cultures. These studies are summarized


in Tables 2 and 3. In the following pages our work on micropropagation,
and the in vitro formation/biosynthesis of secondary metabolites is
discussed.

3 Micropropagation
Mulberry trees are conventionally propagated by grafting and cutting
(Honda 1972). Other propagation methods are seedling and layering.
Layering is carried out by pressing the twigs down into the soil to induce
adventitious root formation. The rooted twigs are then cut off from the
main stem (Hotta 1951). Although most of the Morus species display tremendously strong rooting ability, so that planting stem cuttings directly in the
field suffices to establish a mulberry plantation (Omura 1973), micropropagation has its own advantages (see Bajaj 1992, 1996). Some of the positive
points for raising plants through micropropagation are: large-scale production of true-to-type clones of mulberry, conservation of elite and endangered
germplasm, and overcoming problems of difficult-to-root varieties and
speCIes.

Y.P.S. Bajaj et al.

266

Table 2. Survey of regenerationimicropagation studies on mulberries. (See also Oka and Ohyama

1986; Niino 1995)


Species Ivariety

Explant

Aim of study

Reference

Morus acidosa cv.


Shimaguwa

Apical and axillary


bud meristems

Micropropagation and
preservation

Enomoto (1987)

M. alba. M. bombycis.
M. latifolia,
M. kagayame

Roots. hypocotyls.
young stems, winter
buds, leaves and
shoot tips

Callus cultures, plant


propagation
organogenesis

Ohyama (1970); Ohyama


and Oka (1976); Oka
and Ohyama (1981,
1986)

M. alba

Cotyledons, leaves,
hypocotyls, shoot
tips of embryos

Cloning of genotypes

Kim et al. (1985)

M. alba

Tips of axillary buds

Micropropagation

Hossain et al. (1990)

M. alba

Nodal segments

Micropropagation and
cold storage

Sharma and Thorpe


(1990)

M. alba

Encapsulated adventitious
buds of leaves

Regeneration
of synthetic
seeds

Machii (1992); Machii


and Yamanouchi (1993)

M. bombycis

Calli from internodal


explants

Plant regeneration

Jain and Datta (1992)

M. bombycis cvs.
Kenmochi, Akagi,
Shimizuwase, Ichinose

Meristems of
cryopreserved winter
buds

Plant regeneration and


germplasm preservation

Yakuwa and Oka (1988)

M. bombycis cv.
Kenmochi

Cryopreserved in vitrogrown shoot tips


in alginate beads

Plant regeneration
long-term preservation,
cryopreservation

Niino and Sakai (1992),


Niino (1995)

M. bombycis cv.
Kenmochi; M. indica
cvs. Kanva-2, S-1635,
S-36; M. multicaulis
cv. Goshoerami

Axillary vegetative
buds in calcium alginate
beads

Plant regeneration

Pattnaik et al. (1995)

M. indica

Axillary buds, stem


segments, leaf discs,
petiole explants

Micropropagation

Patel et al. (1983)

M. indica

Leaves and axillary


buds

Plant regeneration

Mhatre et al. (1985)

M. indica

Axillary buds of shoottip cultures in


encapsulated beads

Micropropagation

Bapat et al. (1987)

M. kagayamae

Shoot tip

Plant regeneration

Ohyama and Kawakita


(1975)

M. laevigata

Nodal explants

Propagation of difficultto-root species

Islam et al. (1993)

M. latifolia
M.lhou
M. nigra

Winter buds
Winter bud tips
shoot tips

Plant regeneration
Plant regeneration
Rapid propagation of
local varieties

Oka and Ohyama (1974)


Chang (1985)
Ivanicka (1987)

M. nigra

Shoot tips and nodal


explants

Micropropagation

Yadav et al. (1990)

Morus Species (Mulberry)

267
Table 3. Various compounds isolated/biosynthesised in cell cultures of Morus alba
Compound

Reference

b-sitosterol
Kuwanon 1
Mulberrofuran E, kuwanon Q,R,Y
Kuwanon J,Q,R,V
Mulberrofuran T, kuwanol E
Chalcomoracin, kuwanon J
Artanin
Chalcomaracin, b-sitasterol
Hermiterpene
Prenylchalcanes

Kulkarni et al. (1970)


Ueda et al. (1982)
Ueda et al. (1984)
Ikuta et al. (1986)
Hano et al. (1989a)
Hana et al. (1989b)
Hana et al. (1992a)
Hana et al. (1992b)
Hana et al. (1994a,b)
Hana et al. (1994c,d)

3.1 Explant Preparation and Establishment of Cultures

As explants, we used 1-2-mm apices isolated from shoot tips of actively growing shoots of young self-rooted plant of a local variety of Marus nigra L. The
tips were washed in distilled water and, after removing as many leaves as
possible, they were sterilized in 0.1 % HgCl z for 5 min followed by three washes
in sterile distilled water for 15 min. They were then cultured in test tubes on an
agar solidified medium composed of Knop's macroelements, MS (Murashige
and Shoog 1965) microelements with (mg/l): 0.4 thiamine hydrochloride, 100
mesoinositol, 0.5 BA, 30 g sucrose at pH 5.5. The cultures were incubated at
25C, illuminated by cool white fluorescent tubes at 55 W m -2 with a 16-h day
length.
After 1 month of culture, several small leaves and visible adventitious
buds were formed (Fig. 4A). The regenerated cultures were subcultured on
the same medium but supplemented with 0.1 mg/l GA3 to support adventitious
shoot growth. A little soft, light brown callus was always formed on the basal
cut end of the culture, but it was removed on each transfer to the fresh
medium.
3.2 Shoot Multiplication

The established cultures proliferated into rosettes, and adventitious shoots


were transferred on the basal MS multiplication medium in the presence of
1mg/1 BA, O.lmg/1 lEA, O.lmg/1 GA 3 , and 20g sucrose, at pH 5.3. There was
sufficient multiplication and growth of shoots; however, the cultures were
greenish yellow. Using the same medium in the next subcultures, but replacing
the iron sulfate by FeEDTA at 20mgll, the cultures gradually recovered their
normal green color. Optimal shoot multiplication began from the sixth to
eighth subcultures.
In an effort to suppress basal callusing and to improve shoot multiplication and shoot uniformity, several media and their hormone modifications
were tested. The best growth uniformity seemed to be on the medium of

268

YP.S. Bajaj et al.

Quoirin et al. (1977), but with the formation of thinner and fragile shoots.
However, firm shoots were formed on Snir (1982) medium, probably as a
result of GA3 (0.5 mg/l) in the medium.
3.3 Rooting of Shoots

Plantlets (more than 2cm long) were transferred singly to MS medium with
half-concentration macro elements and full-strength microelements, and supplemented with several combinations of growth hormones. Their effects on
rooting are summarized in Table 4.

Fig. 4A-D. Micropropagation of Moru-s nigra. A Established culture of shoot apex after 1 month
of cultivation in modified Knop's medium. B Multiplied shoots after 1 month on modified MS
medium. C Rooted shoots after 1 month on modidied MS medium with different levels of auxins
fmg/lf. From left to right 1 IBA; 0.2 IBA; 0.2 NAA; 0.2 IBA + 0.2 NAA. D Well-rooted plants 2
months after potting. (Ivanicka 1987)

MaTUS

Species (Mulberry)

269

Fig. 4C-D. Continued

The data show favorable rooting ability in shoots. The first roots
occurred in the 2nd week of culture, and grew without auxin; however,
its presence stimulated root quality and quantity (Fig. 4C). Within the
combinations of auxins tested, O.Smg/l IBA was the best. At a higher IBA
content, callus growth was increased on the basal parts of cultures. A combination of O.2mg IBA and O.2mg/l NAA supported thicker and shorter roots
which were less suitable for transfer, causing a lower survival of shoots in
the soil.

y.P.S. Bajaj et al.

270

Table 4. Effects of hormones on the rooting of shoot" of Morus nigra. (1. Ivanicka)
Hormone
(mg/I)

Rooted shoots/
total shoots

Rooting
(%)

Roots per
shoot

No hormones
0.2 IBA
0.5 IBA
1.0 IBA
0.2NAA
0.2 IBA + 0.2 NAA

14/40
35/59
58/62
32/50
37/54
46/54

35.0
59.3
93.5
64.0
68.5
85.2

2-6
3-5
5-7
3-9
3-7
5-8

Survival in
soil (%)

88.2

" Mean of three replicates.

3.4 Plants Transferred to the GreenhouselField

After 1 month on the rooting medium, the plantlets were set into 80-mm
diameter pots filled with a damp mixture composed of steam-sterilized peat
and agroperlite in the ratio 2: 1. To prevent wilting, each plant was covered
with a glass jar for 10 days. Plants were acclimated for 10 days by lowering
humidity, and finally grown without glasscovers. The hardened plants were
fertilized twice a month with a liquid fertilizer. After 2 months, well-growing
plants were transferred into larger containers filled with a light substrate of
compost, peat, and agroperlite (1: 1 : 1). Survival of potted plants was high, up
to 90%. After 4 months of growth in the greenhouse they were 25cm in length
with true-to-type entire leaves (Fig. 4D).
The leaves of black mulberry varied in shape from entire to lobed, but in
the greenhouse the newly formed leaves of stems grown in the same year were
all entire and true-to-type. However, later, the same plants in the 2nd growth
year were expressively lobed. The lobed leaves persisted on the tree for about
4 years, but then when a tree entered into the mature phase, the entire leaves
began to prevail. The formation of lobed leaves on a tree in vitro is comparable to the same blade shapes on suckers or on summer shoots after thicker
branches have been cut off in an in vivo tree. Therefore, the lobed leaves on an
in vitro tree are not abnormalities, but belong to the usual symptoms of the
juvenile phase of a mulberry tree which persist for some restricted period.
Lobed leaves were also observed on in vitro field plants of M. indica (Patel et
al. 1983).
Semihardwood summer cuttings taken from young in vitro trees of black
mulberry had much higher rooting abilities, up to 90%, in comparison with
those from grafted trees with only 30% rooting. Some observations of the
morphological characteristics of M. nigra in vitro and a grafted tree are given
in Table 5.
In Morus alba after long-term (150 days) incubation of callus tissue
under illumination of about 6000 Ix resulted, shoot formation was observed.
This shoot was transferred to a hormone-free MS agar medium to promote
root growth. When the roots were about 1 cm long, the rooted shoots were
transferred to pots containing vermiculite and sand. Plants bearing at least two
intact leaves were transferred to soil mixed with peat moss and sand. One of

271

Marus Species (Mulberry)

Table 5. Some morphological and physiological measures of


8-year-old Marus nigra local variety of in vitro ad in vivo origin.
(1.Ivanicka)
Measures of organs

In vitro tree

Grafted tree

Shoot length (mm)


thickness (mm)
Leaf length (mm)
width (mm)
Fruit length (mm)
width (mm)
weight (g)
Rooting of summer
cuttings (%)

230
5.1
127
123
2.8
1.9
5.2

278
7.1
138
135
3.1
2.1

90

30

6.1

the plants grown well was planted at the Herbal Garden of Kyoto University.
This micropropagated tree (Fig. 1) has now grown to a height of about
10m, and various secondary metabolites have been studied in it. (Hano et al.
1989c).

4 In Vitro Production of Secondary Metabolites in Morus alba


Numerous investigations have been carried out by S. Ueda and his coworkers
and colleagues on the formation/biosynthesis of various compounds in cell
cultures of Morus alba; their published work is discussed here.
In an earlier study (Ueda et al. 1982), callus and cell suspension produced
mainly CAL and KUJ at high levels. The yield of CAL from the callus tissues
was more than 1000 times that of the leaves infected with Fusarium solani f. sp.
mori. After confirmation of the absence of O-methylated Diels-Alder-type
adduct in the cell cultures, O-methylated precursors including chalcone and
prenylated chalcone derivatives were administered to the M. alba cell cultures.
The cell cultures produced various optically active O-methyl derivatives of
CAL and KUJ by aberrant metabolism. Addition of 2,2' ,4'-trihydroxy4-methoxychalcone yielded 2,2',4' -trihydroxy-4-methoxy-3' -prenylchalcone,
4-0-methylkuwanon J, 4-0-methylkuwanon Q, 4,18/1-di-O-methylkuwanon
J, and 18/1-0-methylchalcomoracin as metabolites. Formation of 2,2'4'trihydroxy-4-methoxy-3'-prenylchalcone indicates that prenylation takes
place after aromatization of the cinnamoyltriketide-derived chalcone skeleton
(Fig. 5). Analogous incubation of the cell cultures with 2'-hydroxy-2,4,4'trimethoxy-3'-prenylchalcone gave an optically active CAL derivative bearing
three methoxy groups at 12/1, 16/1, and 18/1 positions. These O-methylated DielsAlder-type ad ducts are all optically active, having the same stereochemistries
as those of CAL and KUJ, as shown by their idential CD spectra (Hano et al.
1990; Fig. 6). By utilizing the enzyme system of the M. alba cell cultures, the
structure of artonin I, a minor constituent of an Indonesian moraceous plant,

272

y'P.S. Bajaj et al.

07

HO

71

R ~

OHO
R = H: 2,2',4' -trihydroxy4- methoxychalcone
R = prenyl : 2,2',4' - trihydroxy4-methoxy-3' -prenylchalcone

vlf
07

H.

HO

711

CH.

~~Yq;::cPC:
?j IIOR

RO

'.;:

R = H, R' = OH: 4-0-methylkuwanon J


R = H, R' = H : 4-0-=-methylkuwanon Q
R = CH., R' = OH :4.18" - di - Q- methylkuwanon J

~~JCO.
H~.Vo~~ u "~OH

H
2' - hydroxy- 2,4,4' - trimethoxy- 3' - prenylchalcone

r'l

CH.O

"

q
OH
;"
~I
OH

18" - Q- methylchalcomoracin
Fig. 5. Aberrant metabolism of O-methylated precursors in Marus alba cell cultures

A rtocarp us heterophyllus, was established. Namely, administration of


artocarpesin, a major constituent isolated from this plant, to the M. alba cell
cultures resulted in the formation of CAL, KUJ, and artonin I (Hano et al.
1992a; Fig. 7).
Early stages of the biosynthesis of CAL and KUJ was examined by administering [1-BC], [2-13C], and [1,2_B~] acetates to the M. alba cell cultures. The
results indicated that both optically active Diels-Alder-type adducts CAL and
KUJ originate from two molecules of cinnamoylpolyketides (Hano et al.
1989b). The BC-Iabeling pattern in KUJ indicating the incorporation of three
successive acetate units into the ring A and C ruled out the possibility of KUJ
being a member of the retrochalcone (Ayabe and Furuya 1982). The chalcone
skeleton is considered to be formed through the deoxygenation (Ayabe et al.
1988) at C-5 of the cinnamoylpolyketide skeleton, followed by cyclization and
aromatization. The incorporation of 13C-Iabeling in sodium [l_13C]acetate into
the C-3' and 5' positions in ring C of CAL disclosed that the 2-arylbenzofuran
moiety is formed through the cyclization at C-3 and C-8 of the
cinnamoylpolyketide skeleton, followed by decarboxylation. Sodium [1,213Cz]acetate (93 atom % 13C) diluted with an equimolecular amount of
nonlabeled sodium acetate was administered to M. alba cells suspended in
sterile water. After incubation in the dark for 1 week, the cells were harvested
and prepared in a conventional manner to give CAL and KUJ. The BC_

'><
j

300

350

...,..

250

(a)

400

EtOH
II1II

-s

de.

250

r-'}

300

il.

(b)

350

c....
.

...,?t-'"

400

EtOH

p'

450

DIll

Fig. 6. a CD spectra: CAL __ lR"-O-mcthyl-CAL _____ : 4,18"-di-O-methyl-CAL ---------. b CD spectra:


KUJ __ : 4-0-methyl-KUJ _____ : 4-0-methyl-kuwanon Q ---------: 4,18"-di-O-methyl-KUJ ........ .

-10

o I

+5

+10

o I

+10

.1

+20

+30

+40

-.l
W

:::s

"...,...,

0'

'"
~
,:::

""(ii'

""0

C/)

274

YP.S. Bajaj et al.

W
I O~
HO r

... ~I

0 orOH
~I

OHO

artocarpesin

HO

ctY'~O
r
OH

OR

artonin I

3' -preny 1- 2,2' ,4,4' -

tetrahydroxynylchalcone
Fig.7. Determination of the structure of artonin I with the aid of an enzyme system of Morus alba
cell cultures. (Hano et al. 1992a)

/0

3 x malonyl CoA + COAS)

(6)

[~? 1 , HO~:
:/"1
co,
SEnz
}

OH

OH

Fig. 8. 2-Arylbenzofuran skeleton formation in Morus alba cell cultures

labeling pattern of [l,2- 13 C2]acetate-derived CAL also supports the proposed


biosynthesis of 2-arylbenzofuran moiety comprising the aldol-type condensation at C-3 and C-8 of the precursor followed by decarboxylation
(Fig. 8). On the other hand, the chalcone skeleton is formed through the
Claisen-type condensation at C-4 and C-9. Observation of spin-spin coupling
between C-23" and C-24" (J = 42.5Hz) as well as between C-l" and C-6"
(J = 38.9Hz) in [2- 13 C]acetate-derived CAL indicates 13C-Iabeling only on
both carbons of the two starter acetate units in the isoprenoid biosynthesis.
The phenomenon is explainable by considering the participation of the
tricarboxylic acid (TCA) cycle. On administration of [l-13C]acetate, the 13C_
labeling was not incorporated into the prenyl moieties of CAL in spite of the

MaTus Species (Mulberry)

275

regular incorporation of the DC-labeling into the two cinnamoyltriketidederived moieties. Contrary to the satisfactory incorporation of [2-13C]mevalonate into ,B-sitosterol, the BC label from the same precursor was not
incorporated into CAL. [2- 13 C]L-Leucine, a candidate for precursor of
mevalonate, was also not incorporated into the hemiterpene moieties of
CAL, whereas aromatic carbons at 3', 5', 10", 2", and 14", corresponding to
the triketide-derived moieties, were enriched. The labeling pattern from
[2- 13 C]L-Ieucine was the same as that from [l-l3C]acetate. [2-BC]L-Leucine was
thus considered to be metabolized in M. alba cell cultures to [l- 13 C]acetyl
CoA, which subsequently participates in the triketide synthesis (Hano et al.
1992b).
The 100.4 MHz 13C NMR spectrum of CAL obtained by administering [U13C6]D-glucose to M. alba cell cultures demonstrates a satisfactory incorporation of the BC label into all carbons of the molecule. Analysis of the satellite
peaks due to l3e- DC coupling between the component carbons disclosed the
13C-Iabeling pattern in CAL shown in Fig. 9, indicating that this compound
consists of two cinnamoyltriketide-isoprenoid units. However, two independent ways of 13C-labeling were observed in the aromatic rings A and F, which
are formed through the shikimate pathway (Fig. 9). Further analysis of the DC
NMR signals for the shikimate-derived A and F ring carbons revealed that
about 50% of the C-l aldehyde carbon and the penultimate carbon corresponding to erythrose 4-phosphate (E-4P) is disconnected. This C3 + C 1 type
disconnectivity in E-4P-derived moieties may arise through glucose metabolism via two triose phosphates, glyceraldehyde 3-phosphate (GAP) and
dihydroxyacetone phosphate (DHAP), which are equilibrated by triose phosphate isomerase. Both triose phosphates formed from D-[U- 13 Co]glucose
participate in the formation of fructose 1,6-diphosphate (F-1,6P), which
enters into the pentose phosphate cycle via F-6P. glucose 6-P, and then
phosphogluconate. Sedoheptulose-7-phosphate occurring in the cycle affords
E-4P with the 13C-labeling described above. This type of disconnection in the
E-4P-derived moieties was also reported in both shikimate-derived aromatic
rings of the antibiotic, obafluorin (Herbert and Knaggs 1992). The two 13C_
labeling patterns in the A and F rings can be attributed to oxidation of
two different isotopic labeled carbons, pointing to a symmetrical phydroxycinnamoyl intermediate. l3C Label from D-[U-1 3C6]glucose was extensively incorporated into the two hemiterpene moieties (Fig. 9). Further
administration of [1,3- J3 C2]- and [2-J3C]glycerol to the cell cultures revealed a
unique 13C-Iabeling pattern in CAL, as shown in Fig. 9, which suggests a novel
hemiterpene biosynthesis. In the case of the formation of an acetate unit from
the exogenous glycerol by way of glycolysis via GAP and DHAP, [1,3- 13 C2]and [2-J3C]glycerol are converted to [2- 13 C]acetate and [l-l3C]acetate, respectively. The experiment with [1 ,3_13C 2]glycerol revealed the expected enhancements at C-6" and C-24" in the starter acetate units, but the 13C labeling in the
second and third acetate units was reversed, appearing at carbons C-3" and C21". The BC NMR spectrum of CAL obtained from this experiment exhibited
13C_l3C coupling between C-3" and C-4' (Jee = 47Hz) as well as between C-21"
and C-ll" (Jee = 51.4Hz). along with long-range coupling between C-3" and C-

Hhl
and

OH

{'o

{:k:i

OH

Fig. 9. BC-Labeling pattern in CAL from D- [U- 13C6l glucose: .... acetate; CJ-EH) (G-l), pyruvate; _ - - _
erythrose 4-phosphate with a 50% disconnection between the terminal carbon and the penultimate carbon

HO

chalcomoracin

24"

and

t:l

.E .
.E.

V'
to

0\

Marus Species (Mulberry)

277
4

6
. r - ...... r---OH

17"

OH

(a)

21"

(b)

Fig. 10. a 13C-Labeling patterns in CAL from [1,3- IJC,l glycerol. b 13C-Labeling patterns in CAL
from [2- 13C] glycerol

7" (lee = 4.8Hz) as well as between C-21" and C-25" (lee = 4.8Hz). The 13C
enrichments at C-7" and C-25" might be explained by transfer of 13C between
the cis-methyl and the trans-methyl carbons involving the reversible
diene formation. Pulse-feeding experiments with [2- 13 C]acetate which resulted
in a higher incorporation of 13C label into CAL revealed that the transfer
of 13C takes place not only in the isoprenyl unit at C-4' participating in the
[4 + 2]cycloaddition reaction, but also in the other unit remaining intact at C11" (Hano et al. 1992b). A similar phenomenon was also observed in the
experiment with [2-!3C]glycerol (Fig. 10). The !3C NMR spectrum of CAL
obtained in this case exhibited 13e-13C coupling between C-1" and C-2" (lee =
73.4Hz), as well as between C-22" and C-23" (lee = 74.8Hz). These reversed
ways of 13C-Iabeling at the second and third acetate carbons in both experiments imply the participation of the pentose phosphate cycle in the
hemiterpene biosynthesis. The acetyl CoA derived from the resultant GAP
via phosphoglycerate and pyruvate takes part in the hemiterpene biosynthesis as the second and third acetate units. Regarding the origin of the acetate
units participating in the hemiterpene biosynthesis for CAL, it was concluded that the starter acetate unit for the mevalonate synthesis is of
glycolytic origin, while the second and third acetate units originate from
the pentose phosphate cycle (Fig. 11). On the other hand, j3-sitosterol
cooccurring with CAL was biosynthesized in the conventional way (Hano et
al. 1994a,b).
Both [3- 13 C]L-phenylalanine and [3-!3C]L-tyrosine were incorporated intact into the shikimate-derived moities of CAL and KUJ and M. alba cell
cultures (Hano et al. 1994d). Further experiments administering [l-13C]Lphenylalanine and [3-13C]L-tyrosine simultaneously to the cell cultures revealed that the 13C enrichment of a pair of carbons, C-l' and C-8", of CAL
originating from [l- 13 C]phenylalanine and another pair of carbons, C-3 and C-

YP.S. Bajaj et al.

278

sterols & polyketides

glucose

glycolysis

A.A

PO~OH

. /A

H3c....11 COOH

~
~~
~

CH 3 COSCoA
~ f or

1 t acetate
the s

~~o ~ ~~~tooH I~ppC:t.----------'


~I
bH

L----

A
CH 3 COSCoA

for the 2nd and 3rd


acetates

pentose phosphate cycle

HO&CHO
A
Fig. 11. Biosynthetic route to the hemiterpene moieties of CAL in Morus alba cell cultures: .&.
labeling from (1,3- l3C,l glycerol; labeling from [2- l3C] glycerol

CAL

KUJ

Fig. 12. 13C-Labeling patterns of CAL and KUJ in the simultaneous administration experiment
with [1- 13C]-L-phenylalanine (.) and [3- J3C]-L-tyrosine (.&.)

5", originating from [3-13C]L-tyrosine, were 17 and 4%, respectively. In the case
of KUJ, the 13C enrichment of a pair of carbons, C-8" and the chalcone
carbonyl carbon originating from [l- 13 C]L-phenylalanine, and another pair of
carbons, C-/3 and C-5", originating from [3- 13 C]L-tyrosine, were 6 and 1.5%,
respectively. The predominant contribution of [l- 13 C]L-phenylalanine over
that of [3- 13 C]L-tyrosine suggests that both aromatic amino acids contribute in
a parallel way for the biosynthesis of the prenylchalcone derivatives in M. alba
cell cultures and that direct conversion of L-phenylalanine to L-tyrosine is
unlikely (Fig. 12).
Studies were also conducted on dried root bark taken from a
micropropagated mulberry tree, and a new isoprenoid-substituted flavanone,

Marus Species (Mulberry)

279
H

kuwanol C

kuwanol D

Fig. 13. Two new phenolic compounds from the root bark of a mulberry tree redifferentlated
from callus tissues. (Hano et al. 19H9c)

kuwanol C, and a new geranyl-substituted chalcone, kuwanol D, were obtained along with eight known phenolic compounds, morusin, kuwanons U, S,
morachalcone A, 2,2', 4,4' -tetrahydroxychalcone, moracins M, 0, and P
(Hano et al. 1989C; Fig. 13).

5 Extraction and Structure of Intermolecular Diels-Alder-Type


Adducts of Prenylchalcone and Prenylated 2-Arylbenzofuran
Callus tissues of Monts alba subjected to selection gave rise to cell
lines producing characteristic Diels-Alder-type adducts at high levels (Ueda
et al. 1982). Lyophilized callus tissues were extracted with methanol at
room temperature. The extract was concentrated to dryness. The residue
was extracted with acetone. The acetone extract, after concentration to
dryness, was chromatographed on silica gel with chloroform and an increasing
content of acetone as eluents. Each fraction was further subjected to preparative thin layer chromatography (TLC) (silica gel, n-hexane-acetone 1: 1
or chloroform-methanol 6:1) and preparative HPLC (SSC Silica 4251-N,
solvent: ether). Eight Diels-Alder-type adducts, kuwanons J (KUJ) (Ueda et
al. 1982; Ikuta et al. 1986), Q, R, V (Ikuta et al. 1986), mulberrofurans E
(Ueda et al. 1984), mulberrofuran T (Hano et al. 1989a), CAL (Takasugi
et al. 1980; Ueda et al. 1982), and kuwanol E (Hano et al. 1989a), have been
isolated from the callus tissues along with morachalcones A, B, and moracin
C (Takasugi et al. 1978; 1980; Ikuta et al. 1986; Fig. 14). The structures of
KUJ and CAL suggested that the former comes from two molecules of
morachalcone A, but the latter from morachalcone A and moracin C.
From the M. alba callus tissues, all combinations of these monomers,
morachalcone A, -B, and moracin C were isolated. Mulberrofurans F, G, K
(Fukai et al. 1985) and kuwanol A (Hano et al. 1985) isolated from the Marus
root bark are Diels-Alder-type adducts. Mulberrofuran I was isolated from
mulberry root bark. This compound colored red in acidic media (Hano et al.
1984).

\l

OH

"I:

r\l
-

"I:

Q: R,

J (KUJ): R, = R2 = OH
= OH, R2 = H
R: R, = H, R2 = OH
V: R, = R2 = H

OH

OHO

morachalcone B

kuwanon
kuwanon
kuwanon
kuwanon

'JO

~~I

R2

H~W

Fig. 14. Diels-Alder-type adducts and related monomeric compounds in Morus alba cell cultures

morachalcone A

OH

chalcomoracin (CAL) : R, = H, R2 = OH
mulberrofuran T: R, = prenyl, R2 = OH
mulberrofuran E : R, = Rz = H

HO

~"O

~71

R2

OH
H

H~~
OH

moracin C

kuwanol E

00

!'?-

~.
~.

Y'
to

Morus Species (Mulberry)

281

6 Summary and Conclusion


In vitro cultures have been successfully raised from various species of
Morus for plant regeneration, micropropagation, cryopreservation, synthetic
seed production, and for the biosynthesis of a number of secondary
metabolites.
For micropropagation, shoot tips, axillary buds, and nodal segments have
been used. Despite the large differences in the genotypes and explant sources,
it was possible to achieve plant regeneration on relatively simple basal or
modified MS media supporting multiplication and growth of adventitious
shoots, mostly in the presence of 0.5-1 mg/l BA.
It is also important to note that in vitro rooting of shoots on the media with
low auxin content (0.5-1 mg/l IBA or IBA + NAA) was rather easy, and high
in comparison to the well-known difficulties with rooting in vivo cuttings.
Moreover, cuttings of in vitro origin showed much higher rooting abilities than
grafted field trees. This opens the possibility of enlarging the propagation scale
of difficult-to-root genotypes. The regenerated plants, when transferred to the
field, grew into complete trees.
Numerous compounds, such as ,B-sitosterol, kuwanons, mulberrofuran,
chalcomoracin, artonin, hermiterpene, prenylchalcones, etc. have been
biosynthesized/isolated from cell cultures of Marus alba. The cell cultures
produced remarkably high levels of Diels-Alder-type adducts such as CAL
and KUJ. The cell culture system has enabled to establish precise biosynthetic
pathways from primary metabolites to secondary metabolites. For the
biosynthesis of the prenyl moieties of CAL and KUJ, exogenous acetate was
employed after reconstruction of acetate through the TCA cycle and incorporated only into the starter acetate unit for the hydroxymethylglutaryl-CoA
construction. It was further established that the acetate unit for mevalonate
synthesis is of glycolytic origin, while the second and third acetate units originate from the pentose phosphate cycle. The conventional pathway operates
for the biosynthesis of ,B-sitosterol which co occurs with CAL. These facts
suggest that M. alba cell cultures have at least two separate compartments for
mevalonate biosynthesis. Regarding the biosynthesis of cinnamoyltriketidederived moieties, a parallel contribution of L-phenylalanine and L-tyrosine
was observed.

7 Protocol for Micropropagation


7.1 Best Explant

Apices (1-2mm) dissected from shoot trips of actively growing shoots were
the best for establishing culture.

282

y.P.S. Bajaj et al.

7.2 Best Media

Shoot tip cultures established well on a simple medium composed of Knop's


macroelements, MS micro elements in the presence of 0.5 mg/l BA and supplemented substances of the medium.
For shoot multiplication basal MS medium supplemented with I mg/l BA,
O.I-Img/l IBA, O.I-O.Smg/1 GA3, and 20mg/1 of Fe EDTANA2 was used. The
rate of rooting was high and most effective on MS medium at half concentration of macroelements and sucrose, O.Smg/1 IBA, and O.lmg/1 GA 3

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XIV Oenothera Species (Evening Primrose): In Vitro


Regeneration, Production of Flavonoids, Fatty Acids,
and Other Secondary Metabolites
L. SKRZYPCZAK, B. THIEM, and M.

WES~OWSKA

1 Introduction
1.1 The Plant

Species of the genus Oenothera L. (Onagra Miller) from the family


Onagraceae are characteristic of America, the homeland of species acclimated
in Europe (Szafer and Pawtowski 1959; Raven 1968). The American flora has
the most numerous representatives; plants of these species can be found in
natural localities (Rickett 1970, 1971), or they are grown as decorative plants
with white, pink to reddish purple, or mostly bright yellow flowers (Bailey
1950; Encke 1960). A few species are also found in Russia (Shishkin and
Bobrow 1949; Grossgejm 1962). At present, the genus Oenothera is believed to
be distributed throughout the world with the exception of Antarctica
(Rostariski 1992).
The genus Oenothera is divided into 14 sections (Wagner et al. 1985). As
a result of the creation of hybrid forms, pure single-species populations of this
genus are becoming more and more rare. There are two groups of taxonomists, differing in their opinions on its systematics. The total number of
Oenothera species is estimated at 123 by American taxonomists (Raven et al.
1979; Wagner et al. 1985), and at 212 by European authors (Renner 1956;
Rostariski 1965, 1968, 1977, 1985). By 1992, 26 species and permanent hybrids
had been found in Poland (Fig. 1), grouped in three series: Devriesia (3
species), Oenothera (16 species), and Rugglesia (7 species).
The species of the genus in question are herbaceous plants, annual, biennial or perennial, with single leaves, sometimes bipinnated, without stipels.
The radial flowers have four sepals and as many petals, eight stamens in two
whorls, and a stigma divided into four lobes. The fruit is a capsule with
numerous, tiny, hairless seeds (Hegi 1927; Raven 1968). The flowers of some
species are magnificent, usually lemon-colored. Because of the time of day
when the flowers bloom, the species Oenothera biennis L. [Onagra biennis (L.)
Scop.], for example, is called the night candle. The current knowledge of the
genetics of Oenothera has also been published (Harte 1994).

Department of Pharmaceutical Botany, K. Marcinkowski University of Medical Sciences in


Poznan, Wieniawskiego 1, 61-712 Poznan, Poland
Biotechnology in Agriculture and Forestry, Vol. 41
Medicinal and Aromatic Plants X (ed. by Y.P.S. Bajaj)
Springer-Verlag Berlin Heidelberg 1998

Oenothera Species (Evening Primrose)

287

Fig. 1. Ecological plantation of Oenothera paradoxa in liidi-Tuszyn, Poland (Agropharm)

1.2 Importance of Oenothera Species

Species of the genus Oenothera are widely distributed throughout Europe,


They appeared in Europe at the start of the 17th century (Szafer and
Pawrowski 1959), The plant was used as a vegetable, Salads were made from
the roots, which tasted like ham (Madaus 1938), and its seeds served as a
coffee substitute and as bird food. The leaves and roots were used in folk
medicine as remedia metabolica and in homeopathy as antidiarrhoica. Eight
species of Oenothera are mentioned in many pharmacopoeias of the world as
medicinal plants (Pen so 1983).
Nowadays, oil obtained from seeds is very important as a natural dietetic
and biologically active source of unsaturated fatty acids, especially y-linolenic
acid.
Natural drugs are produced by various firms (Sect. 2.2). Oeparol
(Oenothera paradoxa oleum = evening primrose oil) is produced by
Agropharm (Poland). One capsule contains 510mg of oil, cold pressed without
additions of antioxidants or coloring and aromatic compounds.
The drug must meet specific sensorial and physicochemical requirements.
Oeparol has to contain over 73.5% of linoleic acid, and not less than 9% of ylinolenic acid. Moreover, the drug must be free from pollution, and contain
heavy metals only in acceptable amounts (Lamer-Zarawska et al. 1993). On
the basis of the evening primrose oil, the firm Agropharm produces more than
20 different cosmetics and a mosquito-repellent gel.
Horrobin (1990) reported a comparison of biological activity of evening
primrose, fungal, blackcurrant. and borage oils, each containing y-linolenic

288

L. Skrzypczak et al.

acid. It was observed that after the application of the same doses of the oils,
the highest concentration of final metabolites in the blood of rats (e.g.,
prostaglandin PGE j ) appeared after using evening primrose oil.
The medical importance of evening primrose oil, especially of y-linolenic
acid, was described by Horrobin (1990). Positive results are obtained in the
treatment of artheriosclerosis, cardiovascular diseases, atopic eczema, schizophrenia, diabetic neuropathy, multiple sclerosis, Alzheimer's and Raynaud's
diseases, and others. Besides numerous papers on the therapeutic effects of
this oil, some publications report negative results of clinical research (e.g.,
Biagi et al. 1994; Chenoy et al. 1994; Kleijnen 1994).
The aerial part of Oenothera species may also be a source of other biologically active compounds, especially phenolics. Flavonoids with different structures have multidirectional effects on mammals (Middleton and Kandaswami
1994). Other phenol compounds, like phenolic acids (Masquelier and
Delaunay 1965; Wagner 1985) and ellagotannins, exhibit pharmacological
activity. The hydrolyzable tannins show antitumor activity (Miyamoto et al.
1987, 1993a,b; Motoyama et al. 1988; Harborne and Baxer 1993), and also
inhibit HIV replication (As an aka et al. 1988; Nakashima et al. 1992) and
Herpes simplex HSV viruses (Fukuchi et al. 1989). These compounds are
enzyme inhibitors (Kakiuchi et al. 1985; Nishizawa et al. 1989; Kadota et al.
1990; Nonaka et al. 1990) and antioxidants (Su et al. 1988; Okuda et al. 1989),
and they also have antiviral properties (Corthout et al. 1991).

2 Compounds in Oenothera Species


2.1 Phenolic Compounds and Tannins

Among the first reports on flavonoid compounds was one on the presence of
the myricetin 3-,B-galactoside in the whole of the plant o. lavandulaefolia T.
and G. (Kagan 1967), and another on the glucosides of kaempferol and
quercetin found in the flowers and leaves of O. biennis L. (Kowalewski et al.
1968). Comprehensive studies of the American species of the genus Oenothera
were made by Averett et al. (1987, 1988). In the leaves of species from
five sections of the genus: Gauropsis, Hartmannia, Kneiffa, Paradoxus, and
Xylopleurium, they found a total of 33 flavonoid compounds, mostly
glucosides, derivatives of kaempferol, quercetin, isorhamnetin, and myricetin
(Fig. 2). They were the first to discover in the genus Oenothera flavonoid
C-glycosyls and, with the exception of O. speciosa (Averett et al. 1987),
glucuronids and flavonoid sulfates. The major flavonoid of 0. speciosa is
myricetin glucoside (Howard and Mabry 1970).
In the majority of the compounds, the substituents are in the C-3. These
results allowed the authors to engage in chemotaxonomic discussions, supported by their knowledge of the distribution of the species under study
throughout North America. Research was also carried out on Oenothera spe-

289

Oenothera Species (Evening Primrose)

cies of the section Megapterium (Howard et al. 1972; Averett et al. 1991).
Unlike the others, they were found to lack flavonoid sulfates, but, instead, they
had a flavone compound, luteolin 7-0-rutinoside. Chromatographic methods
showed leaves to contain phenolic acids and flavonoids (Szepczyriska and
Wolbis 1992). Other authors identified 16 phenolic acids in 4 species of
Oenothera (Krzaczek and Bogucka-Kocka 1994; Krzaczek et al. 1995).
On the basis of the above works and those by Zinsmeister et al.
(Zinsmeister and Barti 1971; Zinsmeister and Biering 1973; Zinsmeister and
Schels 1975; Zinsmeister et al. 1977), it can be concluded that some 55
Oenothera species have been tested for the presence of phenolic compounds
so far. The biosynthesis of the flavonoids was controlled in seedlings of the

/ \CH/
\CH/ \ (CH )---C~
0 0
2
2 4
2
2 4
'OH

CH- (CH )
3

i-linolenic acid

HO
R

OH

Kaempferol
Quercetin
Isorhamnetin
Myricetin
Luteolin

H
OH
OH
OH
OH

H
H
CH 3
H
H

H
H
H
OH
H

OH
OH
OH
OH
H

Aglycones of flavonoid glycosides

R2

OH

Rl

R2

R3

R4

Vitexin

OH

glucosyl

Isovitexin

OH

glucosyl OH

Orientin

OH

OH

Isoorientin

OH

OH

glucosyl H

C-glycosylflavones
Fig. 2. Main compounds in Oenothera species

glucosyl

290

L. Skrzypczak et al.

HO~
co___o~O--co:g:~::
HO~

&

O-CO

00

HO'"

~OOH

1 CO--:::-<.

""'CO

HO

~
o

HO

'"
..... 1

OH

HO HO

00

OH

O-----Co

"'I
.....

OH

_ _ _ _ CO

OH

CO ~O
.... 0
0
0 __------C

HO

OH

O-CO

OH

OH

OH

:H OH

Oenothein

*OH OH

O~OH

CO/"

OH

~I

CO

'1

'"

~g . .

_____

OH

HOYOH
OH

OH
HO
HO
HO

CO

r&
o

HO
HO

...

/0

-?I

Co~O,

OH

/O~OH

/
CO

0-

HO....OH
OH

HO
~O
0
'\.

/'"

CO----- 0

I",

OH

-CO

OH
OH

*0
CO

............... CO

0,
,

....

...
'"

'"

~:H

Oenothein

OH
OH
OH
OH

HO~OH
OH

Hydrolyzable tannins

Fig. 2. Continued

hybrids of 0. odorata and O. berteriana, and also in O. odorata (Neumann and


Schwemmle 1993, 1994).
As Hegnauer states (1969), whole plants of O. biennis contain an
average of 11 % of tannins. In 0. hookeri T. et G. hydrolyzable tannins
have been shown to occur, 16% in fruits and leaves, about 8% in stems
and flowers, and 2-6% in roots and seeds (Zinsmeister et al. 1965). The
tannins of leaves and stems of 25 species of 7 subgenera of the genus
Oenothera were also investigated in qualitative and quantitative terms.

Oenothera Species (Evening Primrose)

291

The calculated amounts of tannins were 0.9-7.86% for stems and 3.75-17% for
leaves (Zinsmeister et al. 1970; Bartl 1975). Of the four tannin groups
(Hegnauer 1986), noteworthy are the ellagitannins called oenotheins. From
among more than 150 oligomeric hydrolyzable tannins known so far,
oenothein A and B (Fig. 2) have now been isolated as major polyphenols
from the leaves of 0. erythrosepala (Asanaka et al. 1988; Hatano et al. 1989),
O. biennis (Yoshida et al. 1991), and O. laciniata (Yoshida et al. 1995).
Oenothein B was also found in other species from the Onagraceae, Epilobium spp., and Chamaenerion angustifolium (Okuda et al. 1993). These
compounds show biological effects such as antitumor activity (Miyamoto
et al. 1987; Motoyama et al. 1988; Harborne and Baxer 1993). Oenothein
B, with its unique macrocyclic structure, was also produced by partial hydrolysis of cornusiin A (Okuda et al. 1984). From roots and stems
of O. laciniata three new tannins, oenotheins D, F, and G, together
with oenotheins A and B, have been isolated (Yoshida et al. 1995). A minor
trimar, oenothein E, was also isolated. The classification of oligomeric
hydrolyzable tannins and their occurrence in plants was published by Okuda et
al. (1993).
2.2 Oil, Fatty Acids and Other Compounds

Heeger (1948) and Dickmann (1948) described 0. biennis as a vegetable-like


plant, which supplies about 200 kg of oil per hectare. The oil obtained from the
seeds of this species contains 70% of a biologically active linoleic acid, and
10% I"linolenic acid, (gamolenic acid; cis6, cis9, cis12-octadecatrienoic acid;
Harborne and Baxter 1993), only rarely found in plants. The dietetic properties and highly diversified medicinal effects of the oil have aroused much
interest in the Oenothera species. A search for sources of I"linolenic acid has
started; as a result, it has been found to occur in the seeds of Borago officinalis
L. and other species of the family Boraginaceae (Wolf et al. 1983; Sewon and
Tyystjarvi 1993), in the fruits of Ribes nigrum L. and R. uva crispa (Traitler et
al. 1984), in algae (Nichols and Wood 1968; Cohen et al. 1987, 1993), and in
fungi (Shimizu et al. 1988). Those last organisms, e.g., Mucor javanicus, are
used in biotechnological processes of I"linolenic acid production (Ratledge
1991).
In recent years there have been numerous studies concerning the growing
conditions of Oenothera plants (e.g., Levy et al. 1993) as well as works and
patents describing the application and effects of evening primrose oil, especially the I"linolenic acid it contains (e.g., Yaniv et al. 1989). The list of
publications is too vast to quote. A very comprehensive and interesting
study, with a reference list of 467 items, has been published by Horrobin
(1990).
Analyses have also been macJe of the components of evening primrose oil,
discussed in detail by Hudson (1984). Many medicinal and cosmetic preparations use the oil as a base; they are produced worldwide by such firms as
Britannia Health, Horman Chemie, Nattermann, Vitamex, Agropharm, and

L. Skrzypczak et al.

292

Table 1. Summary of in vitro culture studies on Oenothera species


Species

Explant
source

Medium MS Growth
regulators (mg/I)

Response

Reference

O. acerviphilla
RostaIiski
(nomen
provis.)

Seedling
parts
Shoot
tips

2,4-D(I)+ BA(2)

Callus

Our unpub!' results (1993)

BA(I)+IAA(I)

Multiple
shoots

0. biennis L.

Seedling
parts
Seedling
parts
Seedling
parts
Seedling
parts

2,4-D(I)+kin(0.1)
NAA(2)+kin(0.1)
2,4-D+kin(unpub!.)

Callus

Takeo et a!. (1987)

Callus

BA(I)+ IAA(O.I)
BA(I)+NAA(I)
2,4-D(0.25)+ BA(I)

Multiple
shoots
Callus

Osamu and Tadashi


(1987)
Skrzypczak et a!. (1994)

Shoot
tips
Shoot
tips
Callus

NAA(0.2) + BA(0.2)
NAA(0.2)+ BA(2)
IAA(O.I)+ BA(I)
IAA(I) + BA(I)
2,4-D(2), BA(O.5)

Multiple
shoots
Multiple
shoots
Cell
suspension

Shoot
apex

Not mentioned

O. erythrosepala
Borbas

0. hookeri
Torr. et Gray

0. hookeri

Suzuki et a!. (1990)


Our unpub!. results (1993)
Thiem et a!. (1994)
Thiem et a!. (1994)
Wolfson and Sears (1989)

Anthers

2,4-D(2)+ NAA(2)
2,4-D(2)+ BA(2)

Callus
Callus

Martinez and Noher


de Halac (1995)

Seedling
parts
Leaves

2,4-D(0.5), NAA(4),
BA(I)'
NAA(4)+BA(I),
BA(2)"

Callus

Stubbe and Herrmann


(1982)

Shoot
tips
Seedling
parts
Callus

IAA(O.I) + BA(I)
IAA(I)+ BA(I)
2,4-D(2)+ BA(0.5)
Kin(I)+2,4-D(0.5)
BA(0.5)+2,4-D(2)

Multiple
shoots
Callus

Our unpub!. results (1992)


Thiem et a!. (1994)
Our unpub!. results (1995)

Cell
suspension

Thiem et a!. (1994)

Shoot tips

BA(I)+ IAA(I)

Our unpub!. results


(1995)

Seedling
parts

2,4-D(2) + BA(0.5)

Multiple
shoots
Callus

Shoot tips

BA(I)+NAA(I)
BA(2)
BA(I)+ IAA(I)

MUltiple shoots
Multiple shoots
Multiple shoots

Seedling
parts

BA(0.5)+2,4-D(2)
Kin(I)+2,4-D(0.5)

Callus

Our unpub!. results


(1992)
Our unpub!. results
(1995)
Our unpub!' results
(1995)

O. silesiaca
Renner

Shoot tips
Seedling
parts

BA(I) + IAA(I)
2,4-0(0.5) + Kin(l)

Multiple shoots
Callus

O. rubricaulis
Kleb.

Seedling
parts

BA(I)+ IAA(I)

Multiple shoots Our unpubl.results


(1993)

De Vries
O. hookeri

O. paradoxa
Hudziok

O. ammophila
Focke

0. fallax
Renner em
RostaIiski

, Medium of Nagata and Takebe (1971).

Plantlets
Shoots

Our unpub!. results


(1993)

Oenothera Species (Evening Primrose)

293

others. A qualitative analysis of 11 drugs with evening primrose oil produced


by many German firms was carried out by Ihrig and Blume (1994). The species
that has come to be cultivated in Poland is o. paradoxa Hudziok, which is
probably a hybrid of O. silesiaca Renner x O. salicifolia Desf, ex G. Don
(Rostanski 1992). As a new oil plant, it is the basis of today's plantations in
Poland (Fig. 1). Its seeds are used by the Polish firm Agropharm to produce
the preparation Oeparol: doses of oil enclosed in gelatine capsules. Research
is also carried out on the cultivation of Oenothera species and on obtaining
new valuable lines and cultivars of the plants (Lamer-Zarawska and Hojden
1989a,b; Hojden and Lamer-Zarawska 1990; Lamer-Zarawska et al. 1990).
The oil content in the seeds, and the fatty acids in it have been controlled by
Lamer-Zarawska et al. (1989) and Duczmal et al. (1993). The unusual fatty
acid composition of cuticular lipids was examined in leaves of 0. paradoxa
(Jankowski and Storyhwo 1995). In the seed of O. lamarckiana triterpenes
were found (Hopkins and Scheinmann 1971). The materials from the symposia
on Oil from Oenothera in Prophylaxis and Therapy (Symposium 1992, 1995,
bSdf) contain many publications concerning taxonomic, technological, and
analytical issues. Questions of cultivation and results of pharmacological and
clinical tests are also discussed.

3 In Vitro Approaches
3.1 Review
Few studies have been published so far on in vitro cultures of plants whose oil
contains y-linolenic acid (Table 1). Among the first were reports on the formation of organized multicellular structures with 0. coronifera Renner (Jean
et al. 1976), the genome/plastome hybrids from Oenothera (Stubbe and
Herrmann 1982), and patents of Japanese authors (Osamu and Tadashi 1987;
Takeo et al. 1987), who showed that in o. biennis the biosynthesis of this acid
took place already at the stage of callus tissue formation. They grew cultures
in darkness, on MS (Murashige and Skoog 1962) medium enriched with 2,4-D
and kinetin or NAA and kinetin (Table 1). The y-linolenic acid content was
6.7% (Osamu and Tadashi 1987). The stimulus for the initiation and development of the callus tissue from leaves of seedlings of o. johansen L. was
provided by the products of the conjugates of IAA with alanine and IAA with
lysine (Magnus et al. 1992). Suzuki et al. (1990) devoted their studies to in vitro
cultures of O. erythrosepala Borbas, a plant containing oenothein B (2.246.67%), which has antitumor effects. They initiated cultures from shoot tips
on MS media enriched with various concentrations of NAA and BA. The
most numerous buds were observed to develop on media with 0.2 mg/l NAA
and 0.2mg/1 BA, or 0.2mg/1 NAA and 2mg/1 BA. Shoots were rooted on
media enriched with 2mg/1 NAA. The shoot tip cultures proved to be a fast
method of propagation of plants with a high content of active oenothein
B. Wolfson and Sears (1989) also derived cultures from shoot tips of

294

L. Skrzypczak et al.

Table 2. Content of oil and fatty acids in seeds of Oenothera biennis L. (Skrzypczak et al. 1994)

Species
O. biennis L.
from tissue culture plants
O. biennis L.
from in vivo plants

Oil content
(%)

Content of fatty acids (%)


Palmitic

Stearic

Oleic

Linolic

y-Linolenic

24.0

6.59

1.73

10.06

71.92

8.15

22.5

6.54

1.70

10.84

71.53

7.72

O. hookeri, but they did not publish their full results. The report of Martinez
,and Noher de Halac (1995) was the first on in vitro plants developed
from anthers of the genus Oenothera. Research was also carried out on
the micropropagation of O. biennis, with simultaneous control of the oil
content in seeds and fatty acids in the oil (Skrzypczak et al. 1994). Cultures
were initiated from shoot tips or nodal segments on a medium enriched
with various growth regulator concentrations. The most numerous buds
were produced on MS media with BA (1 mg/l) and IAA (0.1 mg/l), or BA
(1 mg/l) and NAA (1 mg/l). On MS medium without growth regulators the
buds developed into shoots after 2-4 weeks; the shoots rooted after 4-7 days
on MS medium containing lEA (0.5mg/I). The culture time of 0. biennis
was about 4 months from seed placement to the transfer of plants into soil.
Callus culture was obtained on MS medium with BA (1 mg/l) and 2,4-D
(0.25 mg/l). The light green callus tissue, often with a pink hue, grew slowly and
did not differentiate. When transferred to cultivation plots, rooted plants
produced a rosette of leaves in the first year, and in the second, bloomed and
produced seeds. Using the gas chromatography method, the oil content in
seeds was determined, and in the oil, the content of fatty acids (Table 2). At
the Department of Pharmaceutical Botany, we have been engaged in the in
vitro culture of O. paradoxa (Table 1) and of other Oenothera species (Thiem
et al. 1994).
3.2 Establishment of Tissue Cultures and Plant Regeneration

3.2.1 Micropropagation of Oenothera Species

In vitro cultures were initiated from the seed collection of the Chair of Biology
and Botany of the Medical Academy in Wroctaw. The seeds of the following
species: O. ammophila Focke, 0. biennis L., 0. erythrosepala Borbas, O. fallax
Renner em Rostanski, 0. paradoxa Hodziok, 0. rubricaulis Kleb, O.
salicifolia Desf ex G. Don, and O. silesiaca Renner, were used.
Special care had to be taken with seed sterilization. The best results were
obtained using a 0.2% sublimate solution (6-7 min) with an addition of Tween80. Seeds free from primary infections and properly sterilized often germinated 100% after 5-14 days.
From 350 to 500 whole, undamaged seeds of each species were placed
on MS medium. The seedlings were used for micropropagation, which
was initiated from shoot tips (6-8mm long) under conditions described pre-

Oenothera Species (Evening Primrose)

295

Fig.3a,b. Tissue cultures of Oenothera paradoxa. a Hypocotyl-derived callus subcultured on MS


medium with 2,4-D (0.25 mg/I) +BA (1 mg/I) in light, 3 weeks after transferring onto a fresh
medium, b Multiple shoots obtained on MS medium with IAA (1 mg/l) and BA (1 mg/I). Growth
period 6 weeks

viously (Skrzypczak et al. 1988). The MS medium was enriched with various
growth regulators. The media used in shoot multiplication and in the induction
and growth of the callus are listed in Table 1. The medium containing IAA
(1 mg/l) was suitable for rooting. Plantlets derived through micropropagation
were transferred into pots, and then to experimental plots. No significant
differences were observed in the morphology and yields of plants derived from
in vitro cultures and regular field cultures. Figures 3 and 4 show some stages in
the micropropagation of the Oenothera species.

L. Skrzypczak et al.

296

O.fM.

Fig. 4a-d. Micropropagation of Oenothera species. a Multiple shoots of O. erythrosepala. b O.


tal/ax obtained on MS medium with IAA (1 mg/I) and BA (1 mg/I). Growth period 6-7 weeks. c O.
ammophila, 3-month-old regenerated plant. d Plants of O. rubricaulis from shoot tip culture in the
1st year of vegetation in the field

Oenothera Species (Evening Primrose)

297

Table 3. Fatty acids in seeds of Oenothera species obtained from culture in vitro

Fatty acids content (%)


Species / year

Palmitic
C 16 : 1

Oleic

Cl8o '

Linoleic
C IU

y-Linolenic
C,u

0. paradoxa Hudziok
1993/94

4.9

5.9

74.8

10.1

1994/95

5.2

6.3

76.9

9.0

1994/95

5.1

5.6

77.4

9.0

1995
O. ammophilla Focke
1995
0. erythrosepala Barbas
1994/95
O. fallax Renner
em Rostariski 1993/94

6.2

4.6

75.9

11.3

6.5

5.3

74.4

11.2

5.1

12.5

71.9

7.7

5.4

10.4

70.1

9.6

O. paradoxa
O. paradoxa
O. paradoxa

3.2.2 Callus and Cell Suspension Induction

Callus tissue cultures were obtained from fragments of seedling-cotyledons,


hypocotyls, or roots (Thiem et al. 1995). In most of the species studied, the best
results were obtained on MS media with BA (1 mg/l) and 2,4-D (0.25 mg/l), or
kinetin (1 mg/l) and 2,4-D (0.5 mg/l).
Callus tissues grown in the light were pale green, with the surface sometimes slightly pinkish (0. ammophila, 0. biennis, O. erythrosepala, O.
paradoxa) or red (0. fallax). Subculturing was necessary every 2-3 weeks.
Callus cultures of 0. paradoxa grown in darkness on MS medium enriched
with 2,4-D (1 mg/l) and kinetin (0.1 mg/l) were colorless. Callus induction and
growth were often made difficult because of the darkening of the tissue (0.
ammophila, O. fallax).
A cell suspension culture was started from a newly developed callus tissue
of O. erythrosepala grown on agar-solidified MS medium enriched with 2,4-D
(1 mg/l) and BA (0.5 mg/l). Some 3-4 g of friable callus was placed in liquid
medium (50cm3 ), incubated in Erlenmeyer flasks (250cm 3 ) in a shaker (at 130
rpm). The cell suspension was subcultured every 10 days by dilution with fresh
medium at a rate of 1: 3. A macroscopic analysis was made to evaluate the
viability of suspension cells.
3.3 Compounds in in Vitro Plants and Callus

To determine the oil contents, and its fatty acids, dry seeds of O. biennis were
used that had been collected from plants grown in vitro. As Table 2 shows, the
results were compared with those obtained for seeds of soil plants cultivated in
the same localities, where plants from in vitro cultures had been grown.

298

L. Skrzypczak et al.

Fig. 5. Chromatogram of butanolic fraction from O. erythrosepala Borbas and standard compounds. G Gallic acid; E Ellagic acid; Cellulose plate, developing phases: I 15% OHAc; II
BuOH(2)-OHAc-H20 14: 1: 5, UV254 after spraying with solution of f3-ethanolamine
diphenylboric acid ester

Encouraged by the information of Japanese authors (Osamu and Tadashi


1987) about substantial amounts of y.linolenic acid found in the callus tissue of
O. biennis, we determined the fatty acid content in material from the species
O. acerviphila. Tissue grown in successive passages was dried at 25C and
extracted with n-hexane in a Soxhlet apparatus for 10h. The extract was
condensed in a vacuum evaporator under reduced pressure at a temperature
below 40C. Fatty acids in the form of methyl esters were determined by
Krzyzaniak and Segiet-Kujawa (1991) using capillary gas chromatography.
They discovered the presence of 0.27% y.linolenic acid, 26.5% a-linolenic
acid, and 15.5% linoleic acid.
Table 3 presents the results of determination of the fatty acid contents in
seeds of various species of the genus Oenothera collected from 1- and 2-yearold plants. The results are means of three determinations carried out with the
help of gas chromatography (GC), after converting the fatty acids into methyl
esters, in the Agropharm laboratory in t6dz-Tuszyn, Poland. The results will
be published together with the analysis of phenolic compounds (Thiem et al.
1998).
In the plants from the in vitro cultures the presence of phenolic
compounds, including ftavonoids and tannins was also studied. As in the
soil plants from America (Averett et al. 1988), the material for analysis
was: calli, multiple shoots, and rosettes of leaves from in vitro cultures of the
species 0. ammophila, O. fallax 0. paradoxa, O. biennis, and O. erythrosepala.

Oenothera Species (Evening Primrose)

299

The material was extracted with methanol three times for 1 h. The extracts
were combined and reduced to dryness under lower pressure at 40C.
Sediments were dissolved in hot water, cooled, and shaken with chloroform
and then butanol. Butanol fractions were reduced to dryness and dissolved
in a few cm3 of methanol. These solutions were subjected to two-dimensional
chromatography (2D-TLC) on Cellulose plates (DC Merck) in phases: I
first direction 15% OHAc, II second direction BuOH(2)-OHAc-HzO
14:1:5 (Bartl 1975). Spots of compounds were analyzed under UV-365
and UV-254 light before and after spraying the plates with 1 % ethanol
solution of aluminum chloride, or 0.1 % ethanol solution of f3-ethanolamine
diphenylboric acid ester (Naturstoffreagenz A, Roth), and a solution of
KIO).
A sample arrangement of the compounds is presented in the
chromatogram (Fig. 5) in leaves of O. erythrosepala from in vitro culture.
The results indicate that the synthesis of phenolic compounds takes place at
the stage of callus and multiple shoots. Only some spots of ftavonoids were
observed on chromatograms of the extracts of callus tissues, whereas other
phenolic compounds also appeared. The extracts from multiple shoots and
leaves showed more than ten ftavonoids on the plates.

4 Conclusion
Seeds of Oenothera species are a source of bio-oil of consumer and medicinal
significance. As a result of our studies with eight Oenothera species, it was
found that micropropagation from both shoot tips and nodal segments follows
a similar pattern. Eight to 14 shoots can be obtained from a single explant. The
cloning of the Oenothera species may find practical application in the case of
some particularly interesting varieties.
The oil synthesized in the seeds of 0. biennis plants derived from
in vitro cultures contains quantities of fatty acids similar to those in the
seeds of soil plants (Table 2). The results are especially interesting for
the contents of the C-18: 3 acid in the seeds of 1-year-old plants of O.
paradoxa and 0. ammophila in comparison with its contents in the
seeds of 2-year-old plants (Table 3). Osamu and Tadashi (1987) had earlier
reported y-linolenic acid (6.7%) in the callus tissue of the species
mentioned.
Apart from y-linolenic acid with its diverse pharmacological effects, noteworthy is the occurrence of ftavonoids and oenotheins belonging to the
ellagotannin group with anticarcinogenic and antiviral properties which have
been found in the species 0. erythrosepala, 0. biennis, and 0. laciniata. Preliminary analyses using 2D-TLC indicate the presence of ftavonoids, ellagic
and gallic acids, and probably their derivatives (Fig. 5) in callus, multiple
shoots, and leaves.
The results of our studies and those of others show that the method of
micropropagation from existing meristems can be used in mass production of

300

L. Skrzypczak et al.

the Oenothera species while preserving their genetic stability (Bajaj et al.
1988).

5 Protocol
5.1 Micropropagation
Stratified seeds, surface sterilized (6-7 min) in 0.2% HgCl, with two drops of Tween-80.
Explants: Shoot tips (6-8mm long) of seedlings (30 days old).
Medium: Shoot multiplication on MS medium with IAA and BA (Table 1).
Culture conditions: 16-h photo periods (2000 Ix) at 25C, relative humidity 60-80%.

5.2 Callus and Cell Suspension Induction


Explants: different parts of seedlings-callus culture.
Medium: MS basal medium supplemented with: kinetin and 2,4-D or BA and 2,4-D (see
Table 1).
Suspension induction: 3-4 g soft, friable callus transferred into 50 cm 3 MS medium with 2,4D and BA in 250-cm 3 flasks and shaken at 130rpm.
Subculture every 10 days in proportion 1 : 3 of fresh medium.
Culture conditions: as above.

5.3 Chemical Analysis


Oil and fatty acids were determined in the seeds (Sect. 3.3) using Gc. Phenolic compounds in
butanol fractions were examined by 2D-TLC on plate with Cellulose (DC, Merck). Solvents (v/v):
15% OHAc-first direction I, BuOH(2)-OHAc-H,O 14: 1: 5 in second direction II.
Chromatograms were analyzed under UV-365 and UV-254 before and after spraying with
ethanolic: 1 % AICI 3, 0.1 % J3-aminoethanol ester of diphenylboric acid (Naturstoffreagenz A,
Roth) and KJ0 3 (Harborne 1991).
Acknoledgments. Part of this project was supported by grant No.6 P206 001 of the Committee for
Scientific Research (KBN) for B.T. The authors are grateful to Mrs Mariola Pawlik for her
assistance in the preparation of this manuscript.

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XV Otacanthus Species: In Vitro Culture, Plant


Propagation, and the Production of Essential Oil 1
Ae. RONSE 2 , H. DE POOTER', A VAN DE VYVER2 , and M.P. DE PROFT4

1 Introduction
The genus Otacanthus (family Scrophulariaceae) comprises six species that are
distributed in east Brazil. They are half-shrubs with stems lignified at the basis,
and opposite, lanceolate to obovate leaves; the inflorescence is a terminal
spike of solitary flowers with a green calyx and blue to purple, bilabiate corolla
with a white spot on the lower lip. All species possess ornamental value due to
their showy flowers that last for several weeks, especially Otacanthus coeruleus
Lind., which has the largest flowers (Fig. 1). This species originates from east
Brazil, in the states Rio de Janeiro and Espirito Santo, where it is sometimes
called incenso, which means incense. It is also found cultivated or naturalized
in north Brazil and on the Mascarenes and Seychelles (Ronse 1993). It was
cultivated as an ornamental in Belgium and France during the last century
(Van Houtte 1862), and is now again being cultivated as a pot plant in several
countries. It has also potential as a cut flower crop (Geertsen 1990).
The aerial parts of O. coeruleus contain considerable amounts of an
essential oil that consists of mono- and sesquiterpenoids (De Pooter et al.
1989). The composition of this oil is interesting from a phytochemical point of
view, since j3-copaene-4a-ol, a sesquiterpenoid that was identified for the first
time, is one of its major components (Fig. 2). Another important component
is a-copaene, which is used as a feromone in the control of Ceratitis capitata,
the Mediterranean fruitfly (Jacobson et al. 1987). It constitutes 3 to 5% of the
essential oil.
Other species of Otacanthus were found to contain essential oils consisting
of mono- and sesquiterpenoids in relatively large amounts as well. The species
that were analyzed are 0. coeruleus, O. jluminensis, 0. platychilus, 0. villosus
(three morphologically and chemically different forms of the latter), an unnamed species, and intermediate taxa between these species. Some of them
contained large amounts of unidentified sesquiterpenoids (Ae. Ronse et al.

I Dedicated to the memory of Prof. Dr. H. De Pooter, who passed away in December 1995
, National Botanic Garden. Domein van Boechout, B-1860 Meise, Belgium
3 Formerly: Laboratory for Organic Chemistry. Faculty of Agronomy, University of Gent.
Coupure Links 653. B 9000 Gent. Belgium
4 Laboratory for Plant Physiology, Faculty of Agronomy. Catholic University of Leuven, De
Croylaan 42, B 3001 Heverlee, Belgium

Biotechnology in Agriculture and Forestry, Vol. 41


Medicinal and Aromatic Plants X (cd. by y.P.S. Bajaj)
Springer-Verlag Berlin Heidelberg 1998

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A.C. Ronse et al.

Fig. 1. Flowering plant of Otacanthus coeruleus

Fig. 2. Structure of ,B-copaen-4a-ol

1997a, in prep.). The content and composition of these essential oils is of value
for the taxonomy within the genus, especially for the identification of taxa with
intermediate characters, that are probably hybrids. Furthermore, the presence
of significant amounts of essential oils within several genera of the Scrophulariaceae gives indications for the classification at subfamily level (Ronse
1993).
Conventional propagation of Otacanthus can be done either by taking
cuttings or by sowing. Stem cuttings taken in February and treated with a
commercial rooting preparation (0.5% IAA) all rooted when placed in peat on

307

Otacanthus Species

a heated greenhouse bench. They were grown in a greenhouse with minimum


temperature of 18 cC, and yielded flowering plants within 3 to 6 months,
depending on the species (A.e. Ronse, unpub. results). Seeds are obtained by
self-pollination and germinate within 1 to 2 weeks in the warm greenhouse.

2 Volatile Constituents of the Intact Plant


2.1 Structure and Distribution of Secretory Structures

The structures that secrete essential oils in vegetative parts of plants can be
either trichomes, oil cells, or cavities and ducts (Fahn 1979). In order to find
out the parts of Otacanthus where essential oil is located, freeze-microtome
sections were stained with Sudan Black B and viewed with a light microscope

Fig. 3. Scanning electron micrographs of the


glandular trichomes of Otacanthus coeruleus.
(Ronse 1993). 1 Type 1 hair on abaxial side of
young leaf, X2500; 2 type 2 hair on inner side
of calyx, X2500; 3 hairs of type 3 on petiole,
X160

308

A.C. Ronse et al.

(Ronse 1993). This showed the essential oils to be situated in three types of
glandular trichomes at the surface of the aerial plant parts. Photographs of
these trichomes taken with scanning electron microscopy are shown in Fig. 3.
The first type is a capitate glandular hair with a short unicellular stalk bearing
a round head of four cells (Fig. 3, photo 1); it has a length of 48 pm. The second
type (Fig. 3, photo 2) is very similar but has a head consisting of two cells, and
an average length of 34 pm. The third type (Fig. 3, photo 3) has one or two
stalk cells, one neck cell, and one head cell, with a total length of 77 pm on
average, which becomes three times as large on adult leaves. Trichomes of
type 1 are present only on stems and leaves, those of type 2 on leaves and the
calyx, and type 3 occurs on stems, leaves, the calyx, and the outer surface of the
corolla.
2.2 Content and Composition of the Essential Oil

The essential oil content of the aerial parts of Otacanthus coeruleus amounts to
0.2 to 0.3% in fresh weight (De Pooter et al. 1989). The bulk of this is produced
by the leaves, but the stems have a considerably lower content (0.02 %); some
oil is also produced by the flowers, especially the calyx (0.19%, vs. 0.03% in the
corolla; Ronse 1993).
The volatile oil is a complex mixture of up to 64 mono- and sesquiterpenoid compounds. The composition of the essential oil found in intact
plants at different stages of maturity is shown in Table 1. Compounds that
amount to more than 1 % of the oil are listed here, yielding a total of 25
constituents. The sesquiterpenoids with Kovats index larger than 1580 were
found only in nonflowering shoots of older plants, albeit in small amounts. Of
the 28 identified monoterpenoids, 50% are oxygenated compounds, while of
the 12 identified sesquiterpenoids only 17% are oxygenated. The quantitatively most important compounds are the monoterpenoids trans-pinocarveol,
pinocarvone, myrtenal, and myrtenol, all belonging to the pinane type, and the
sesquiterpenoids f3-copaen-4a-ol and a-humulene; together they constitute
about 50% of the total oil.

3 In Vitro Culture Studies


In vitro techniques have been applied for callus production and for plant
regeneration by axillary shoot production of o. coeruleus, 0. platych ilus , and
O. villosus (Ronse and De Proft 1992; Ronse et al. 1997b). This work is
reviewed in Section 3.1, while the further text deals with the production of
volatile oils by in vitro cultures of O. coeruleus.

Otacanthus Species

309
Table 1. Composition of the essential oil of aerial parts of
Otacanthus coeruleus

Peak
no.

Ip"

2
4
5
11
17
18

929
965
969
1021
1086
1093

19
20

1108
1126

21
22
23
24
25
26
27
28
29
34
37
47
48
49
52
54
61

1131
1141
1155
1166
1173
1176
1182
1200
1207
1374
1451
1577
1582
1594
1617
1656
1756

Compound

Peak area (%)


Ab

a-Pinene
Sabinene
,B-Pinene
Limonene
Linalo01
tr-Sabinene
hydr.
Monoterpenoid
Transpinocarveol
Camphor
Pinocarvone
Isopinocamphone
Terpinen-4-01
Myrtenal
a-Terpineol
Myrtenol
Cis-carveol
Trans-carveol
a-Copaene
a-Humulene
~-Copaen-4a-ol

Sesquiterpenoid
Sesquiterpenoid
Sesquiterpenoid
Sesquiterpenoid
Sesquiterpenoid

4.6
4.0
2.8
0.9
1.0
1.8

4.4
1.8
2.3
0.9
1.0
1.3

4.2
2.6
2.7
0.9
1.8

1.6
1.9
1.3
1.0
0.8
1.5

1.5
11.6

1.6
13.4

1.5
13.1

1.0
13.7

0.4
11.3
0.6
1.1
7.3
1.0
4.3
0.1
3.0
4.6
7.3
8.3

0.3
12.3
0.7
1.7
7.2
0.8
6.0
1.0

1.6
12.1
1.2
1.8
6.7
1.4
5.8
1.7
1.7
2.8
5.2
6.0

1.0
6.5
0.1
0.9
5.8

1.1

3.2
6.6
7.7

1.1

6.0
1.0
1.2
3.8
6.7
13.0
2.5
3.6
4.5
2.5
2.1

" Ip Kovats index. b A 2-month-old plants; B young plants before


blooming; C shoots after blooming; D nonflowering shoots of
older plants. (A, B, and C after De Pooter et al. 1989; Dafter
Ronse 1993.)

3.1 In Vitro Propagation

The basal medium for micropropagation of O. coeruleus consists of the


macroelements and iron-EDTA solution of Murashige and Skoog (1962), the
micro elements and vitamins of Nitsch and Nitsch (1969), 6 gil agar, and 25 gil
sucrose (Ronse and De Proft 1992). Leaf pieces and nodal stem segments were
put on this medium with several combinations of plant hormones. As a result
all leaf explants died except with 5.0pM NAA + 2.2pM BA, where callus was
formed. However, nearly all stem explants responded to the treatments by
callus formation, outgrowth of nodal buds or shoot proliferation (Table 2; Fig.
4). Callus was obtained from stem segments with several combinations of
auxins and cytokinins, the combination of 5 pM NAA + 2.2 pM BA yielding
the highest number of callus and the fastest-growing callus. Formation of
adventitious buds and shoot proliferation was obtained from stem segments

A.C. Ronse et al.

310

Table 2. Organogenic response of nodal stem segments of Otacanthus coeruleus to various types

and concentrations of plant growth regulators (in % of explants). (Ronse and De Proft 1992)
Plant growth
regulators

Callus
formation

CuM)

R1

NAAO.5
NAA 0.5
NAA 0.5
NAA 0.5
NAA 5.0
BA2.2
BA 13.2
2,4-D 0.5
2,4-D 2.5

0
0
43
6
100
88
0
0
37
0

+ BA 0.44
+ BA 2.2
+ BA 4.4
+ BA 2.2

Outgrowth of
nodal buds

Shoot
proliferation'

R2

R1

R2

R1

R2

0
50
86
50
17
100
43
0
43
100

9
43
43
24
0
0
0
0
5
0

12
75
86
20
0
0
43
0
43
0

0
0
43
35
0
0
0
0
0
0

0
25
86
80
67
29
57
0
29
0

The results of two replications are given, consisting of 12 and 10 explants, respectively.
R1 and R2: percentage of explants with a particular response in replication 1 and 2, respectively.
" Shoots formed from adventitious buds.

A,B
Fig. 4A-D. Nodal stem segments grown for 3 months on the basal medium supplemented with
different plant growth regulators (in .uM). A 2.2 BA + 0.5 NAA. B 0.5 NAA. C 0.5 2,4-D. D 0.44
BA + 0.5 NAA

with 0.5,uM NAA + 0.44 to 4.4,uM BA. The most rapid proliferation was
observed with 0.5,uM NAA + 2.2,uM BA, yielding a proliferation rate of 6
every 2 weeks. For a rapid micropropagation of 0. coeruleus, this treatment
was used to produce shoots that were subsequently rooted on hormone-free

Otacanthus Species

311

medium with 1 gil charcoal. Shoots could also be obtained from calli placed on
Monnier's medium (Monnier 1976). In vitro-produced plantlets were transplanted in the greenhouse with 80% survival rate, and flowered 10 months
later.
Several environmental factors were found to influence the growth velocity
and the morphology of the shoot cultures of O. coeruleus (Ronse et al. 1997c).
The factors tested were the temperature (day/night 25120 resp. 20/15 0C), the
irradiance level (either 30 or 55 pmollm2/s at plant level), the agar concentration (from 0 to 10 gil), the macrosalt concentration (from 114 to 2 times the MS
concentration) and sucrose concentration (from 5 to 50 gil); the sucrose treatments were given either without or with mannitol addition so as to obtain an
osmotic pressure of -3.5 bar.
Growth increased both with temperature and irradiance, and was optimal
with 6 gil agar, with between 20 and 30 gil sucrose, and at a macrosalt concentration equal to 1 or 1.5 times that of Murashige and Skoog medium. Significant mortality occurred at the lowest and highest sucrose concentrations,
except when mannitol was added. The addition of mannitol also enhanced
growth at most sucrose levels. The morphology and appearance of the cultures
was not influenced at the tested temperatures and irradiance levels, but it was
clearly influenced by the agar and by the sucrose concentration, and especially
by the macrosalt concentration. Agar concentrations under 6 gil yielded fastgrowing, thin, and vitrified shoots. A similar but stronger effect was observed
at the two lowest macrosalt concentrations, while the remaining macrosalt
treatments yielded slower proliferating shoots with better-developed leaves
(Fig. 5). Low sucrose concentrations, either without or with mannitol, also
gave vitrified, but highly proliferating explants.
Two other species of Otacanthus were also propagated in vitro.
Otacanthus platychilus was micropropagated successfully from nodal stem
segments that formed adventitious buds on the basal medium supplemented
with 0.5 pM NAA + either 2.2 or 4.4 pM BA. The highest cytokinin concentration gave the fastest proliferation of the explants, but many were vitrified and
died. With 2.2 pM BA, only a fourfold multiplication of the explants after 2
months was found, but these were healthy. Leaves of this species only responded to a hormone treatment of 5 pM NAA + 2.2 pM BA, which yielded
callus. Shoots were rooted with 100% success after a few weeks on hormonefree medium with 1 gil charcoal. Plantlets were transplanted to the greenhouse, where they began flowering after 16 weeks.
Nodal stem segments of Otacanthus villosus died on the basal medium
supplemented with 0.5 pM NAA + 4.4 pM BA, but they formed adventitious
buds and proliferating shoots with 0.5 pM NAA + 2.2 pM BA; the latter
treatment gave 3.5 times the number of initial explants after 5 weeks. Shoots
were better rooted on hormone-free basal medium without charcoal, than with
1 gil charcoal.

312

A.C. Ronse et al.

Fig. SA-D. Proliferating shoot cultures of O. coeruleus on macrosalt concentration 25-200% MS;
(see text)

3.2 Extraction Methods and GCIMS Analysis of Essential Oils

Essential oils were extracted from plant tissue cultures and intact plants by
means of a microsteam distillation apparatus (Chrompack), according to the
method of Likens and Nickerson (1964), and adapted to small quantities (170 g) of plant material. The plant material was cut and placed in the flask with
distilled water and boiling chips for regular boiling. The solvent used was
dichloromethane. The water flask was surrounded by an oil bath with a temperature of 120-150 C, while the solvent flask was put in water of about 70C.
The extractions were effected during 2h. After drying the extract over MgS0 4,
the solvent was evaporated from the extract in a light vacuum. The extracts
were kept in a deep freezer under nitrogen atmosphere.
The extraction of essential oils from nutrient media was effected by mixing the nutrient media with distilled water, and then shaking this mixture in
a separating funnel with a mixture of two thirds pentane and one third
dichloromethane. The extraction was repeated four times consecutively with
each nutrient medium, and the obtained solvent fractions were put together.
This fraction was washed with a 5% NaCl solution. After drying over MgS04
for several hours and after filtration, the solvent was evaporated in a light
vacuum.
The analysis of the components of the essential oils was made primarily
by gas chromatography with a Varian 3700 instrument equipped either with

Otacanthus Species

313

home-made SE-30 glass capillary columns (40 or 60m X 0.5mm i.d.; coating
thickness 0.75 ,urn) or with a FSOT RSL-150 capillary column (25 m X 0.53 mm
i.d.; coating thickness 1.2,um), and an FID. Injector and detector temperatures: 220C; oven temperature: programmed from 30 to 220 C at 3C/min;
sample: 0.5,u1 of a solution of 0.5,u1 oil in 50,u1 pentane; on-column injection;
carrier gas: 3.65 ml He/min. Peak areas were calculated by a PDP 11/34
computer. Kovats indices (Ips) were obtained by coinjection of a solution of
the homologous hydrocarbon series C6-C18 with the sample solution in a
temperature-programmed run, and linear interpolation of the location of
the peaks.
Mass spectra were obtained after gas chromatography in a Varian 2700
instrument equipped with an SE-30 FSOT capillary column (25m X 0.53mm
i.d.; coating thickness 1.2,um), coupled to a Varian MAT 112 mass spectrometer by means of a platinum capillary (i.d. 0.133 mm), or with an HP 5890 gas
chromatograph equipped with an 80-m FSOT capillary column (50m SE-30 on
the injector side + 30m SE-52 X 0.2mm i.d.; coating thickness 0.2,um) and an
HP 5970A mass selective detector.
Substances were identified on the basis of their Ips and mass spectra,
which were compared with those of reference compounds. These were purchased, isolated from or identified in essential oils of known composition.
Components with a difference of Ip of 4 can be distinguished from each other.
The mass spectra are compared with known spectra of compounds by an HP
9133 workstation with GC-MS-MSD operating software HP 59974A (series
200-GCMS Master). The program uses the ten peaks with the highest intensity, and weights them by using the product of the intensity times the mass, so
that ions with a high mass become more important. For identification, we
accepted a correlation down to 98%, but in all cases the mass spectra were
controlled visually with those from the reference library. Moreover, the Ip was
used as control for the identity of the peak. Sometimes, mass spectra with
correlations as low as 75% turned out to be well identified.
3.3 Volatile Components in the Cultures of Otacanthus coeruleus
3.3.1 Oil Content
We found that all types of plant tissue cultures of 0. coeruleus contained an
essential oil under all environmental conditions, though their content varied
strongly, from 0.001 to 0.063% in fresh weight. The main factor influencing the
oil content proved to be the type of culture, as well as its growth rate (Table 3).
The growth rate, as indicated in this table, reflects the average total weight and
proliferation rate of the explants after 3 months, the latter being defined as the
number of explants obtained from one original explant. The highest essential
oil content was found in cultures that produce both callus and shoots, and
amounts to about 10% of the content in intact plants. Furthermore, a high
growth rate influenced the oil content negatively, at least in proliferating shoot
cultures.

314

AC. Ronse et al.

Table 3. Essential oil content and number of constituents of the oil in tissue cultures of different
type and growth rate
PGR treatment
CuM)

Culture
type"
C

2.52,4-D
5.0 NAA
0.5NAA
0.5 NAA
0.5 NAA
0.52,4-D
0.5 NAA
2.2BA

+ 2.2 BA
+ 4.4 BA
+ 2.2 BA
+ 0.44 BA

+
+

+
+
+

Growth
rateb

Oil
content
(%)

No. of
components

S
F
S
F
FF
S
S
S

0.004
0.009
0.014
0.007
0.001
0.026
0.028
0.022

43
42
43
35
44
23
56
51

+
+
+
+
+
+

" Culture type: C callus; S formation of axillary shoots.


b Growth rate: S slow; F fast; FF very fast (see text for definition).

The oil content of fast-proliferating cultures (PGR treatment of 0.5 11M


NAA + 2.2mM BA) was examined in relation to the number of days in vitro,
and in relation to several micro-environmental conditions. This showed that
neither the number of days in vitro, nor the macrosalt concentration of the
medium affected the oil content with a clear trend, and random fluctuations
between 0.001 and 0.037% were measured. The oil yield increased with the
sucrose concentration of the medium, mainly due to the increase in fresh
weight, but also due to a higher relative oil content at the highest sucrose
concentration (50g/l). A similar effect of sucrose was observed at a constant
osmotic pressure, suggesting that it can be ascribed to the energy effect of
sucrose. Increasing agar contents of the medium resulted in higher oil contents
of the cultures (Ronse 1993).
3.3.2 Oil Composition

The culture type influenced the composition of the essential oil, as indicated by
the number of components of the essential oil (Table 3). This number lay
between 23 and 56, which is smaller than in the intact plant. Analysis of the
identity of the compounds showed that the essential oils that most resemble
that of the intact plant were found in the shoot cultures and in some of the
mixed cultures (cultures with shoots and callus). The callus cultures and some
of the mixed cultures produced oils that are less similar to that of the intact
plant, as illustrated in Fig. 6. Callus obtained with 2.5 11M 2,4-D contained the
same constituents as in the intact plant, but in different percentages, e.g., it
contained more sesquiterpenoids.
In all types of cultures, the otherwise important sequiterpenoids
,B-copaen-4a-ol and a-humulene were lacking or were present only in
small amounts. Other compounds occurred in completely different
amounts than in the intact plants under several PGR treatments. The

Otacanthus Species

315

47

A
37

27
34

2 4

I ~ II 17 It
_J._JLlLJ.~~

! 25.

L~

~ ~W
27

n.......-rrrn,.-".-"
II
37

20
17

22

52

48

54

Time (min)
I I I I

I I I

I I I I
10

I I I I

20

I I I I I I I I I
30

I I I I

I I I I
40

I I I I I I
50

Fig. 6. Gas chromatogram of the essential oil of nonflowering shoots of O. coeruleus (A) and
of a callus culture obtained with 2.5,uM 2,4-D (8). Numbering refers to the peak numbers in
Table 1

sesquiterpenoid valencene, for example, increased dramatically in content


(from a trace up to 29%) in cultures treated with a combination of
0.5.uM NAA and with 0.44 to 4.4.uM BA. In the same cases, the sesquiterpenoid a-copaene was also present in higher quantities. Another
striking example is an unknown sesquiterpenoid with Kovats Index of
approximately 1900, that was not detected in intact plants, but was present

I I I I I

316

A.C. Ronse et al.

in important amounts (up to 39%) when only auxins, either 2,4-D or NAA,
were added to the cultures.
The sucrose, macrosalt, and agar concentration of the medium influenced
the relative content of the constituents. Some substances, such as valencene,
showed a large variation without apparent relation with the factors studied.
Other substances, such as the monoterpenoids of the pinane type, were
present in constant quantities after one subculture, but after five subcultures of
3 weeks, their content had significantly decreased at low sucrose and low
macrosalt concentrations, and increased at standard and high sucrose concentrations. High osmotic pressure obtained by mannitol addition, on the contrary, caused an increase in these compounds at low sucrose concentration
after five subcultures.
3.4 Volatile Components in the Nutrient Media

3.4.1 Oil Content


From all nutrient media that were examined, we extracted a significant
amount of essential oil. This ranged between 0.14 and 2.28mg per test tube. In
many cases, more oil was extracted from the nutrient medium than from the
cultures growing on it. In proliferating shoot cultures the total oil production
per fresh weight (in mg/g) decreased by more than half between 1 and 3 weeks
of culture, and it remained at a lower level during further culturing. This
decrease was due to an increase in fresh weight with time, but also to a lower
oil content in the medium.
High sucrose treatments increased the oil quantities found in the medium,
especially from 40 gil on, as shown in Fig. 7a. A similar increase was shown for
the relative oil content in mg oillg fresh weight (Fig. 7b). It is also obvious that
there was an increase from 20 gil on in the proportion of oil found in the
medium.
3.4.2 Oil Composition
The composition of the essential oils extracted from the nutrient media
was different from that of the cultures themselves, but was as stable.
An example of it is given in Table 4, showing the composition of an oil
recovered from a nutrient medium on which proliferating shoot cultures
had been growing for 31 days. In total, 25 compounds have been listed that
repeatedly occur in oils extracted from nutrient media; of these, 21 often
constitute more than 1 % of the oil. The quantitatively most important compounds are unidentified sesquiterpenoids with Kovats index of resp. 1914,
1400, 1825, 1955, and isomenthol. These substances have not been found
in the plant cultures; only four compounds have also been identified in
the explants, namely l3-caryophyllene, valencene, J3-copaene-4a-ol, and
allo-aromadendrene.

I-

0.'

10

20.

10

20.

I I I

30.

40.

I I I

sucrose concentration (in gil)

I I I

I I I

50.

I I I

~
-I

MEDIUM

PLANT

2.5

3.0

"'a

-0

CL

Qj

l'

co

0.

0.5

1.0

1.5

l' 2.0

:c

OJ

"E

0,

3.5

5.

20..

30..

40..
sucrose concentration (in gill

10.

50..

Fig.7A,B. Essential oil content of in vitro cultures and nutrient media in relation to sucrose treatment. A Oil per treatment (in
mg)_ B Total oil content per fresh weight (in mg/g)

u
0

C 30.
0

<D

S 40.

0,
E

50.

60.

70.

----l

D:

o.

(0

VJ
'0

1:;

s:.

;,

Ei

A.c. Ronse et al.

318

Table 4. Composition of the essential oil extracted from a nutrient medium with proliferating
shoots of 0. coeruleus after 31 days of culture
Ip

Compound

Ip

1131
1140
1157
1172
1277
1351
1388
1400
1420
1459
1488

Menthon
Isomenthon
Menthol
Isomenthol
Menthylacetate

0.8
0.2
0.3
2.3
0.7
3.9
0.2
12.0
0.7
0.2
0.2

1511
1549
1580
1600
1655
1700
1825
1914
1932
1955

j3-Bourbonene
j3-Caryophyllene
Valencene

Compound

j3-Copaene-4a-ol

1.7
1.1
0.3
1.7
1.8
1.0
14.6
30.8
1.2
4.0

4 Conclusion and Prospects


Otacanthus coeruleus is fairly easily micropropagated from stem explants by
adventive bud formation; several combinations of plant growth regulators
produce callus and/or shoot cultures. Otacanthus platychilus and O. villosus
were also micropropagated successfully from stem segments with nearly the
same treatments as for 0. coeruleus. Considerable amounts of essential oils
have been recovered from in vitro cultures, not only from the plant cultures,
but also from the nutrient media on which they had grown.
The oils extracted from the shoot and callus cultures contained approximately the same mono- and sesquiterpenoids as the intact plants, but in
different relative quantities. Moreover, the number of substances was reduced
by 12 to 64 % in comparison to that of the plants, which still gives a complex
mixture. The cultures contained up to 0.06% essential oil in fresh weight,
which represents about one third of the content in plants. This is more than
what has been reported from other similar studies.
The oil found in the nutrient medium also contained mono- and
sesquiterpenoids, and the composition was relatively stable in all circumstances. Most constituents, however, are substances other than in the plants,
which could possibly be explained by a diffusion of some substances from the
cultures in the medium, followed by chemical transformation. Surprisingly, the
amount of essential oil extracted from the nutrient media was very high, often
higher than the amount extracted from the cultures. The total oil content per
fresh weight (in cultures plus medium) sometimes even reached 0.34%, which
is more than in the plants! Reports of the occurrence of essential oils in solid
nutrient media are rare, and mostly small quantities have been found. In many
studies, however, this has not been investigated. A possible explanation of our
results is that essential oil compounds are diffused or secreted into the medium
as a detoxification mechanism. We have, indeed, found that a higher proportion of essential oil is present in the medium at high sucrose concentration,
when the plant cultures are stressed and do not grow optimally. This

Otacanthus Species

319

hypothesis, however, requires additional observations, with an analysis of the


presence of glycosylated compounds.
Acknowledgments. The authors thank Marcel Verhaegen for handling the scanning electron
microscope and taking the micrographs, as well as for making Fig. 6.

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VI. Springer, Berlin Heidelberg New York, pp 252-263
Ronse A, Van de Vyver A, De Proft M (1997c) Effect of environmental factors on in vitro growth
and morphology of Otacanthus coeruleus (Scrophulariaceae). Belg J Bot 130 (1) (in press)
Van Houtte L (1962) Otacanthus coeruleus. Flore Serres Jard Eur 15:53-54

XVI Oxalis Species: In Vitro Culture,


Micropropagation, and the Formation of
Anthocyanins
J.

VAN STADEN

1 Introduction
The genus Oxalis L. is the largest of the Oxalidaceae (Cronquist 1981;
Mabberley 1987), and is represented by some 500 species worldwide. While
being cosmopolitan, the main centres of diversity for Oxalis are in South
America and South Africa. The South Western Cape of the latter region is
particularly rich in Oxalis species; Salter (1944) reported 208 species. These
dicotyledonous plants exhibit a wide range of growth habits (herbs, many
geophytes with bulbs or tubers, undershrubs) and inflorescence structures
(Salter 1944). Their leaves are alternate, usually compound, digitate or pinnate. Most species exhibit nastic movement. In addition to having attractive
shapes, most spectacular during rainy months, leaves of some species also have
attractive spotting. Flowers are regular, bisexual with a pentamerous perianth.
Under bright light conditions, the flowers of Oxalis open in a wide range of
hues, varying from white through yellow to scarlet. Bicoloured tubular or bellshaped flowers occur, mostly with yellow throats (Salter 1944). Many Oxalis
species are geophytes and bear a variety of perennating organs, including root
tubers, stolons, bulbili and bulbs. Aerial bulbili are also produced by some
species. Seeds are small, endospermous and are produced in a fruit (capsule)
the dehiscence of which is explosive (Knuth 1930).
The genus is of considerable economic importance and this will increase
with realization of the ornamental potential of many of the small herbaceous
species (Table 1). Their flowering characteristics (Fig. 1) make them suitable
for horticultural development and introduction as small bedding plants. One
problem that may be experienced in this process is the fact that some Oxalis
species have become naturalized outside their normal habitat. As a result of
their propensity for bulbil formation, some species have become troublesome
weeds (Holt 1987 Marshall 1987) which may harbour inoculum for diseases
(Roos and Hattingh 1986). However, many species have a highly localized
distribution, suggesting that not all taxa have this weedy tendency. Some
species, such as 0. cernua, contain oxalate in levels toxic to livestock (Rekhis
1994). 0. erosa has been mentioned as a medicinal species (Arenas 1981). In

Natal University Research Unit for Plant Growth and Development, Department of Botany,
University of Natal Pietermaritzburg, Private Bag X01, Scottsville 3209, South Africa
Biotechnology in Agriculture and Forestry, Vol. 41
Medicinal and Aromatic Plants X (ed. by Y.P.S. Bajaj)
Springer-Verlag Berlin Heidelberg 1998

Oxalis Species

321

Table 1. List of Oxalis species studied for economic reasons


Oxalis species

Reason studied

Reference

O. cernua Thunb.
O. corniculata L.
O. dispar N .E. SR.

Rekhis (1994)
Holt (1987)
Sunderland (1966)
Sunderland and Wells (1968)
Ochatt and De Azkue (1984)

O. glaucifolia Knuth.
O. gracilis Jacq.

Oxalate poisoning
Weed, germination
Plastid and pigment
development
Ornamental and medicinal
plant
Ornamental plant
Ornamental plant

0. hedysaroides H.B.K.
O. helicoides Salter

Ornamental plant
Ornamental plant

O. latifolia H.B.K.
0. linearis Jacq.
0. pes-cap rae L.

Weed
Anthocyanin production
Source of inoculum for
bacterial canker
Weed
Anthocyanin production
Ornamental plant

O. erosa Knuth.

O. reclinata Jacq.
O.
O.
0.
O.

regnellii Miq.
rhombeo-ovata A. St. Hil.
stricta L.
tuberosa Mol.

O. variifolia Steud.

Ornamental, viral disease


Ornamental plant
Weed
Ornamental crop
Vegetable crop
Diseased, mycoplasm-like
bodies
Ornamental plant

Ochatt et al. (1988)


Crouch and Van Staden
(1994)
Maene and Debergh (1981)
Crouch and Van Staden
(1994)
Marshall (1987)
Meyer and Van Staden (1995)
Roos and Hattingh (1986)
Marshall (1987)
Crouch et al. (1993)
Crouch and Van Staden
(1994)
Coyier (1981)
Ochatt et al. (1988)
Marshall (1987)
Ochatt et al. (1986)
Khan et al. (1988)
Atkey and Brunt (1982)
Crouch and Van Staden
(1994)

Fig.I. Dense-flowering habit of oxalis helicoides in winter while growing in a greenhouse (Crouch
and of Van Staden 1994)

322

1. Van Staden

South America, O. tuberosa (oca) is an important food crop, second only to


the potato in the high Andes (Rea and Morales 1980). This species has been
accepted as a vegetable and ornamental outside its natural habitat in temperate regions (Khan et al. 1988).
Apart from the above economic uses, Oxalis species have been used for
scientific studies into pigment and plastid development (Sunderland 1966,
1967; Sunderland and Wells 1968). Recently, it was shown that red callus
from 0. reclinata contains cyanidin-3-glucoside (Crouch et al. 1993). Attempts
were made to optimize the production of this anthocyanin in callus of O.
linearis (Meyer and Van Staden 1995) as it can serve as an important natural
colorant.

2 In Vitro Culture Studies


Table 1 contains a list of Oxalis species that have been studied for various
economic reasons. As early as 1966, O. dispar was grown in culture, not
for micropropagation, but to investigate plastid and pigment development (Sunderland 1966). Subsequently, members of the genus were tissue
cultured for three main reasons: development of ornamental and food
potentials, and for anthocyanin production (Table 1). The results of these
tissue culture studies are summarized in Table 2. In vitro cultures of Oxalis
spp. have been successfully established using shoot tips, stem nodes, stem
internodes and tuber cores. Noticeable is that there are no reports where bulbs
or bulbili served as explants. Sodium hypochlorite, with or without a
surfactant, was the sterilant most frequently used for decontamination of
explants (Table 2). In the case of O. hedysaroides stem node explants, a
combination of alcohol, HgC1 2 and NaOCl was used effectively (Maene and
Debergh 1981). In all cases, sterilization was followed by exhaustive rinsing
with sterile distilled water. Following disinfestation, explants have been
cultured most successfully on MS medium (Murashige and Skoog 1962)
containing 3% sucrose, 0.6-0.8% agar, supplemented with various combinations of a-naphthalene acetic acid (NAA) and 6-benzyladenine (BA) or
kinetin, adjusted to pH 5.7-5.8 before autoclaving. Cultures were generally
maintained under cool white fluorescent lights at a 16-h light 8-h dark cycle at
25 3C.
2.1 Micropropagation

Seed production in Oxalis is prolific in most species. Germination of fresh seed


occurs over a broad yet seasonally dependent range of temperatures (Holt
1987). The plants apparently hybridize readily, providing a means for horticultural improvement. The array of perennating organs (tubers, stolons, bulbili,
bulbs) produced increases the reproductive potential of many species.

0. glaucifolia

0. g/aucifolia

0('

4. Red callus

pH 5.7
0.1 NAA + 1 kinetin
+1 GA,

Complete plants

Red callus

MS.
a.Bult) agar.

3. Cell colonies

2 NAA + 0.1 kinetin

Cell colonies

0.5 2.4-0 + 2 kinetin

B5 liquid or
0.5%
agarose

2. Protoplasts from
internodal callus

1. Stem internodes

Callus

Rooted plantlcts

Shoots

Callus

Rooted plantlcts

Shoots with
expanded k'ave:-.
and elongated
internodes

Adventitious shoot
huds

2 NAA + 0.1 kinetin

pH 5.7

16/8 h light/dark.
irradiance I.B Wm
24 + 1 0 ( ,

25

Green. nodular
callus

Response

MS
O.H% agar.
pH 5.7

2.4lyo NaOCl+O.1 %
Tween-20. 10 min

0.1 CiA, filter


sterilized or
autoclaved

Liquid MS on
Heller
hridges.

3. Shoots

2 NAA + 0.1 kinetin

0.5 kinetin + 0.1


GAl

pH 5.7

MS.
O.B% agar.

2. Callus

1. Stem
internodes

2.4(% NaOCl+O.1 (Xl


Tween-20. 10 min

0.005 IBA

Li4Uid 1/2
MS

4. Elongated

shoots

O.9GA,

5.~

16/8 h light/dark.
irradiance I.B Wm

11.1 IBA + 0.1 GA, +


11.5 BA

3. Adventitious
shoot huds

pH

MS.
O.B% agar.

Culture conditions

Growth regulators
(mg/I)

11.5 BA + 0.1 GA,

1.2% NaOCI+II.I%
Triton X-IOO.
ISmin

I. Shoot tips

O. erosa

Basal medium

2. Nodular callus

Sterilization

Explant source

Oxalis species

Table 2. Summary of Oxalis tissue culture research

Ochatt et al.
(1989)

Ochatt el al.
(1988)

Ochalt and
De Azkue (1984)

Reference

W
N
W

(')

'~."

."

'"Vl

~;::.,

Stem internodes

1. Stem nodes

O. gracilis

O. hedysaroides

MS,
0.8% agar.
pH 5.7

1.75% NaOCI,
4 min

Stem internodes

O. reclinata

2 NAA + 0.1 kinetin


or
5 NAA + 0.5 BA

5 NAA + 0.5 BA

MS,
0.8% agar.
pH 5.7

1.75% NaOCl,
4min

Stem internodes

O. reclinata

0.4 NAA + 1.8 BA

MS,
0.2 % Gelrite,
pH 5.7

0.2% HgCI"
10 min

Stem internodes

O. linearis

2 NAA + (U kinetin
or
5 NAA + 0.5 BA

MS,
0.8% agar,
pH 5.7

1.75% NaOCI,
4 min

Stem internodes

3 IAA + 3 kinetin

MS,
0.6% agar,
pH 5,8

Alcohol dip,
0.5% HgCl z, 3 min,
8% NaOCI+
Teepo!, 15 min
0,5 kinetin + 0,5 BA

2 NAA + 0,1 kinetin


or
5 NAA + 0,5 BA

MS,
0.8% agar,
pH 5,7

I. 75 % NaOCI,
4min

Growth regulators
(mgll)

Basal medium

Sterilization

O. helicoides

2. Stem with two


nodes

Explant source

Oxalis species

Table 2. Continued

16/8 h light/dark,
irradiance 44.5 .umol
m-1s 1.25 3e
or
21.1 {fmol m- 2 s- 1
10 2C

16/8 h light/dark,
irradiance 44.5 .umol
m-1s 1,25:!::3C

+ roots +

+ roots +

+ roots +

Callus
shoots

Shoots
bulbs

Red callus,
anthocyanin
production

Red callus.
anthocyanin
production

Shoots
bulbs

or
21.1.u mol m- 2 s l ,
10 :!: 2C

Callus + roots

Shoot elongation,
2,5 em long
rootable cuttings.

shoots

Growth of existing
buds and axillary
branching of new

shoots

Continuous light
5000 Ix

+ roots

Shoots + roots +
bulbs

Callus

Response

16/8 h light/dark,
irradiance 44.5 .umal

m-: S-I, 25 3 C

Continuous light.
21,umolm 2S"I,
23 :!: 2C

16/8 h light/dark,
irradiance 44.5 ,umol
m- 2 s- l ; 25 3C
or
21.1,umolm 2S"I,
10 2C

Culture conditions

Crouch and
Van Staden (1994)

Crouch ct al.
(1993)

Meyer and
Van Staden (1995)

Crouch and
Van Staden (1994)

Maene and
Debergh (1981)

Crouch and
Van Staden (1994)

Reference

V-J

'"

(1)

Q..

[/)

'Oi"

,...,

.j:>.

Tuber cores

Stern internodes

0. variifolia

2. Regenerated
shoots

Apical stem
internodes

4. Protoplastderived callu~

1.75% NaOCI.
4min

Tuber peeled,
sterile cork horer

0.8% agar.
pH 5.7

MS.

pH 5.8

MS.
0.8% agar,

MS.
0.8% agar,
pH 5.7

3. Cell colonies

0.6% NaOCI.
20min

agar,
pH 5.7

MS.
O.~%

B5 liquid or
0.5%
ag,uose

2.4% NaOel,
0.1 (Yo Tween-20.
lO min

2. Protoplasts from
internodal callus

1. Stem internodes

Liquid MS on
Heller
bridges.
pH 5.7

2 NAA
or
5 J';AA

+ 0.5 BA

+ 0.2 kinetin

3 NAA + 3 zeatin

or

3 NAA + 3 BA

21.1umolm -s
]():': 2C

or

16/8 h light/dark.
irradiance 44.5 pmol
m ~ s I, 25 ::!::: :; C

Callus + shoots

3 NAA + 3 zeatin
or
3 NAA + 3 BA

Shoots
bulbs

roots +

Callus + roots

Callus

Rooted plantlets

Vascular nodules

0.1 NAA + 1 kinetin


+1 GA,
16/8 h light/dark.
irradiance 30 !imol
m C s 1,25 C

Protoplast -derived
callus

Callus

5 NAA + 0.5 BA

BA

Rooted plantlets

Shoob

Callus

Cell colonies

+ 0.5

irradiance 1.8 Wm-':>


24:': I 'C

161X h light/dark.

0.5 2A-D -I- 2 kinetin

5 NAA

OJ GA, filter
sterilized or
aUloc!aveu

5 NAA + 0.5 BA

0.5 BA + 0.1 GA,

MS.
O.K% agar,
pH 5.7

2. Callus

10 min

2.4% NaOCI+
D,l % Tween-20.

3. Shoots

1. Stem internodes

0. tllberosa

O. tllberosa

O. rhombeo-ov{tfa

0. rhomheo-ovata

Table 2. Continued

Crouch and
Van Staden (1994)

Khan et al. (19KX)

Khan ct aJ. (1988)

Ochatt ct al. (19RL))

(19RR)

Ochatt et al.

W
N

Ul

OJ>

(')

'"
r;;'

-0

(jJ

'~"

;?

326

1. Van Staden

However, bulbil production varies greatly within the genus and many species
are threatened in the wild (Crouch and Van Staden 1994). In O. hedysaroides,
an attractive pot plant, regeneration by cuttings is low due to the poor regeneration capacity of mother plants (Maene and Debergh 1981). Thus tissue
culture holds potential for rapid multiplication of economically desirable
species.
In developing the food potential of Oxalis species it will be necessary to select for reduced oxalate content, tubers with high specific
gravity and increased frost tolerance (Khan et al. 1988). In vitro techniques could help in understanding the morphogenetic potential of
various tissue explants and in identifying conditions for shoot regeneration. Khan et al. (1988) reported morphogenetic (vigour, leaf shape, tuber
shape and size, pigmentation, internode length) and compositional
(chromosome number, sugar and oxalic acid content) variation in in
vitro-generated O. tuberosa plants. This suggests a considerable degree
of somaclonal variation. While undesirable in the micro propagation
process, such somaclonal variation may be beneficial in plant improvement
programmes.
Indications are that Oxalis species are infected with viruses (Coyier 1981),
mycoplasm-like bodies (Atkey and Brunt 1982), and that they serve as a
potential source of inoculum for bacterial canker of stone fruit (Roos and
Hattingh 1986). In vitro culture of small meristematic regions or shoot tips
may help in eradication and control of various diseases, thus improving plant
quality.
2.1.1 Axillary Bud Proliferation

Bud proliferation was studied using stem node explants of O. hedysaroides (Maene and Debergh 1981) and I-mm shoot tips (comprising
the meristematic dome + three leaf primordia) of O. erosa (Ochatt and
De Azkue 1984). Stock plants for these studies were grown in a greenhouse.
With 0. hedysaroides the existing buds elongated and branched optimally
on an IAA- and kinetin-containing medium (Maene and Debergh 1981).
Using a combination of 0.5 mg/l kinetin and 0.5 mg/l BA, shoot proliferation and elongation was achieved to produce rootable cuttings (2.5 cm
long).
The shoot-tip procedure used for O. erosa was more elaborate and timeconsuming (Ochatt and De Azkue 1984). The axillary buds did not elongate
but produced a green nodular callus. Nodular calli were induced to develop
adventitious shoot buds using BA and gibberellic acid (GA3). Application of
0.9mg/C GA3 stimulated internode elongation and leaf expansion. The individual shoots were rooted in half-strength MS containing 0.005 mg/l indole
butyric acid (IBA).

Oxalis Species

327

2.1.2 Shoot Regeneration from Callus Cultures

The majority of tissue culture systems reported for Oxalis spp. used stem
internodes for the initiation of callus, subsequently manipulated in a series of
steps to produce plantlets (Table 2). In the case of 0. tuberosa, callus was
produced from tuber sections but no organogenesis was observed (Khan et al.
1988). Ochatt et al. (1988) developed a three-phase system for 0. glaucifolia
and 0. rhombeo-ovata where internodal callus generated on NAA and
cytokinins was subsequently induced to form shoots using the cytokinins
kinetin or BA plus GA,. The shoots were successfully rooted using halfstrength liquid MS medium containing 0.1 mg/l GA 3 In O. glaucifolia the
callus changed colour during subculture and turned red. Organized structures
were only produced consistently from green callus. Regeneration potential
diminished and was erratic with heterogeneously coloured or red callus
(Ochatt et al. 1988).
Limited callus formation followed by organogenesis was observed in other
studies (Khan et al. 1988; Crouch and Van Staden 1994). A microscopic study
using O. tube rosa indicated that calli first gave rise to thick roots. Swellings
arose at the base of these roots, which may well have been root tubers, as
they were tapered in shape. These swellings then developed into single or
multiple shoots (Khan et al. 1988). Combinations of 3 mg/l NAA with 3 mg/l
BA or zeatin gave best shoot formation, the latter cytokinin being most
effective.
In four South African species with great horticultural potential, callus
formation was rapid and 70% of all internodal explants produced callus within

Fig.2. Organogenesis in O. reclinata following transfer of cultures from 25 to 10C under constant
cool white fluorescent light (21 pmal m- 2 Sl) Arrows indicate bulbs. (Crouch et al. 1993)

328

1. Van Staden

21 days (Crouch and Van Staden 1994). As observed for O. tuberosa (Khan et
al. 1988), root formation initially was more prolific than shoot production.
Transfer of callus of all four species from a growth condition at 25C to a cold
room at 10 2C and a lower light intensity (21,umolm- 2 S-I) resulted in
extensive organogenesis. Shoots, roots, and, most importantly, bulbs developed (Fig. 2). Roots again developed before shoots. Both auxins and
cytokinins were necessary for organ development (Table 2). At lOoC and a
low light intensity, the vitality of developing plantlets improved. After repeated subculture flowering occurred under these growth conditions (Crouch
and Van Staden 1994; Fig. 3).
2.1.3 Plant Regeneration via Pro top lasts

Ochatt et al. (1989) cultured protoplasts obtained from stem internodal


callus of O. glaucifolia and O. rhombeo-ovata, and observed cell colonies
which subsequently produced red or green callus, respectively. 0. glaucifolia

Fig.3. In vitro flowering (arrows) of O. helicoides at lOoC in constant cool white fluorescent light
(21.umol m- 2 S-I). Cultures were maintained on 5 mg I-I NAA and 0.5 mg r l BA. (Crouch and
Van Staden 1994)

Oxalis Species

329

plantiets were regenerated from the red callus produced from the protoplasts. In other Oxalis callus systems the morphogenetic potential was
lost when the callus became pigmented, even in the presence of auxins
and cytokinins (Ochatt and De Azkue 1984; Ochatt et al. 1988; Meyer and
Van Staden 1995). Why the protoplast-derived red callus behaved differently is unknown. In the case of 0. rhombeo-ovata, protoplasts produced only a nodular green callus that did not develop into plant lets.
The availability of a protoplast technique will be useful for somatic hybridization and in overcoming natural barriers for sexual crossing within the
genus.

3 Anthocyanin Production
In a number of instances, Oxalis cultures turned red during culture (Khan et al.
1988; Ochatt et al. 1988, 1989; Crouch et al. 1993). The pigments were isolated
by crushing callus and extracting three times with 3% TFA-MeOH for 24h at
5C. The MeOH concentrate was dissolved in H 2 0 and partitioned against
EtOAC. The aqueous phase was extracted with n-butanol, which was concentrated to yield the crude extract. The crude extract and pigment were never
heated above 30C. The major pigment in 0. reclinata internode callus cultures (Fig. 4) was identified by NMR and GC-MS as cyanidin-3-glucoside

Fig.4. Anthocyanin-rich callus line of O. reclinata, maintained at 25C in the light (16h) provided
by cool white florescent tubes (44.5 pmol m -2 S-'). Cultures were subcultured every 3 weeks on 2%
Gelrite. (Crouch et at. 1993)

J. Van Staden

330
5'

HO

OH

OH

OH
Fig. 5. Structure of cyanidin-3-g1ucoside.

(Crouch et al. 1993; Fig. 5). Anthocyanin production was more prolific at 25
than at 10 C, which favoured organogenesis. It is also produced in suspension
cultures (Crouch et al. 1993). As this anthocyanin could serve as a natural
colorant its production in vitro was examined in stem internodal callus of O.
linearis (Meyer and Van Staden 1995). Anthocyanin-containing callus can
grow in the absence of cytokinins, but not without auxins. Callus growth was
improved with the addition of 8 or 32,uM NAA. The addition of 8,uM BA to
media containing 8 or 32,uM NAA inhibited callus growth. Growth regulators
had a different effect on anthocyanin production. Highest anthocyanin production occurred at a combination of 8,uM BA and 2,uM NAA (Table 3). Of
various cytokinins tested, iso-pentenyladenine (iP), significantly stimulated
callus growth. Pigment production was increased by BA, thidiazuron (TZ) and
zeatin (Table 4). Elevated levels of sucrose (300 to 360mM) stimulated pigment production but inhibited callus growth. Callus growth was best at 60mM
sucrose (Table 5).

4 Protocol
Surface sterilize stem internodal explants with sodium hypochlorite. Place
explants on MS medium containing 2-5 mg/l NAA and 0.1 mg/l kinetin or
0.5 mg/l BA solidified with 0.8% agar. Maintain cultures at 25C under cool
white fluorescent tubes under a 16-h light 8-h dark cycle. Transfer to 10C
induces shoots, roots and bulbs.

Oxalis Species

331

Table 3. The effect of BA and NAA on the growth and


anthocyanin production in Oxalis linearis callus. (Meyer and
Van Staden 1995, with kind permission of Kluwer Academic
Publishers)
Growth regulators
(uM)
BA

NAA

0
0
0
0
k
k
k
k

0
2
k
32
0
2
k
32

Growth
index

Specific
absorbance

0.1
1.2
6.Ob"
6.4b
0.5
0.7
4.la
3.9a

1.71
U5a
0.64a
0.42a
2.47b
2.k6c
2.26b
2.13b

" Treatments with different symbols are significantly different at a


95% level of confidence.

Table 4. The effect of cytokinins on the growth and anthocyanin


production of Oxalis linearis callus cultured in basal medium supplemented with 8,uM NAA. (Meyer and Van Staden 1995, with
kind permission of Kluwer Academic Publishers)
Cytokinin
(8,uM)

Growth
index

Specific
absorbance

Control
BA
Kinetin
iP
TZ
Zeatin

5.9
2.6b"
4.6
7.7a
3.6
1.9b

1.24
2.19a
1.65
1.13
2.l0a
2.95a

" Treatments with different symbols are significantly different at a


95% level of confidence.

Table 5. The effect of sucrose on the growth and anthocyanin


production of Oxalis linearis callus. (Meyer and Van Staden 1995,
with kind permission or Kluwer Academic Publishers)
Sucrose
(mM)

Growth
index

Specific
absorbance

60
120
IkO
240
300
360

4.4
3.5
3.3
3.0
2.0a
1.5a

O.k2
1.36"
1.56a
1.7ka
3.03b
3.2kb

, Treatments with different symbols are significantly different at a


95% level of confidence.

332

1. Van Staden: Oxalis Species

References
Arenas P (1981) Etnobotanica Lengua, Maskoy-FECIC, Buenos Aires
Atkey PT, Brunt AA (1982) The occurrence of mycoplasma-like bodies in severely diseased oca
(Oxalis tuberosa) plants from Bolivia. Phytopathol Z 103:294-300
Brummitt RK (1992) Vascular plant families and genera. Royal Botanic Gardens, Kew
Coyier DL (1981) Chlorotic ringspot and decline of ornamental shamock (Oxalis regnellii). Plant
Dis 65:275-276
Cronquist A (1981) An integrated system of classification of flowering plants. Columbia University Press, New York, pp 826-828
Crouch NR, Van Staden J (1994) In vitro propagation of a number of South African Oxalis
species. S Afr J Bot 60:134-135
Crouch NR, Van Staden LF, Van Staden J, Drewes FE, Drewes SE, Meyer HJ (1993) Accumulation of cyanidin-3-glucoside in callus and cell cultures of Oxalis reclinata. J Plant Physiol
142:109-111
Holt JS (1987) Factors affecting germination in greenhouse-produced seeds of Oxalis corniculata,
a perennial weed. Am J Bot 74:429-436
Khan MRI, Heyes JK, Cohen D (1988) Plant regeneration from oca (Oxalis tuberosa M.): the
effect of explant type and culture media. Plant Cell Tissue Organ Cult 14:41-50
Knuth R (1930) Oxalidaceae. In: Engler A (ed) Das Pflanzenreich 95. Facsimile Edition (1966)
Engelmann-Cramer, Weinheim, pp 1-389
MabberJey DJ (1987) The plant book. Cambridge University Press (1993 reprint), pp 420-421
Maene L, Debergh P (1981) In vitro propagation and culture of Oxalis hedysaroides H.B.K. cv.
Fire Tree. Med Fac Landbouw Rijskuniv Gent 46/4:1201-1203
Marshall G (1987) A review of the biology and control of selected weed species in the genus
Oxalis: O. stricta L., O. lati/olia H.B.K. and O. pes-caprae L. Crop Protection 6:355-364
Meyer HJ, Van Staden J (1995) The in vitro production of a anthocyanin from callus culture of
Oxalis linearis. Plant Cell Tissue Organ Cult 40:55-58
Murashige T, Skoog F (1962) A revised medium for rapid growth and bioassays with tobacco
tissue cultures. Physiol Plant 15:473-497
Ochatt SJ, De Azkue D (1984) Callus proliferation and plant recovery with Oxalis erosa Knuth
shoot tip cultures. J Plant Physiol117:143-146
Ochatt SJ, Ciai AA, Caso OH (1986) Tissue culture techniques applied to American crops:
micropropagation of Oca (Oxalis tuberosa Mol.), an Andean tuber-bearing species. Turrialba
36:187-190
Ochatt SJ, Stampacchio ML, Escandon AS, Martinez AJ (1988) Plant regeneration from callus of
two shrubby Oxalis species from South America. Phyton 48:21-26
Ochatt SJ, Escandon AS, Martinez AJ (1989) Isolation, culture, and plant regeneration of
pro top lasts of two shrubby Oxalis species from South America. J Exp Bot 40:493-496
Rea J, Morales D (1980) Catalogo de Tuberculos Andinos. Inst Boliviano de Tecnologfa
Agropecuaria, Bogota
Rekhis J (1994) The poisonous plant Oxalis cernua. Vet Hum Toxicol 36:23
Roos IMM, Hattingh MJ (1986) Weeds in orchards as potential source of inoculum for bacterial
canker in stone fruit. Phytophylactica 18:5-8
Salter TM (1944) The genus Oxalis in South Africa. A taxonomic revision. J S Afr Bot Supp Vol
1
Sunderland N (1966) Pigmented plant tissues in culture. I. Auxins and pigmentation in
chlorophyllous tissues. Ann Bot 30:353-368
Sunderland N (1967) Pigmented plant tissues in culture. II. Growth, development, and decline of
chlorophyllous tissues. Ann Bot 31:573-591
Sunderland N, Wells B (1968) Plastid structure and development in green callus tissues of Oxalis
dispar. Ann Bot 32:327-346

XVII Polypodium vulgare L. (Wood Fern): In Vitro


Cultures and the Production of Phytoecdysteroids

1 Introduction
The wood fern Polyp odium vulgare L. (Fig. 1) belongs to the family Polypodiaceae, and is the most widely distributed fern species in the world, appearing mostly in warm, humid forests (Bonnier 1934). The name Polyp odium
arises from the particular shape of its rhizomes, branching like feet. The plant
size ranges from 10 to 50cm; the fronds have a very long petiole and the blade
is deeply divided in 20 to 40 alternate lobules. The large sori are present almost
through entire year and are placed on two parallel lines among the median
vein on the underside of the lobes. The penducled sporangia open transversely
throughout a longitudinal ring called the annulus (Strasburber et aL 1983). It
is a very popular fern in Europe and some purgative and vermifugal properties
are attributed to its rhizomes (Vol1ik et aL 1990).
Ecdysteroids (Fig. 2) are the molting hormones of insects and crustaceans. Since ponasterone A was discovered in plants in 1966 by Nakanishi
et aI., over 100 ecdysteroid structures (named phytoecdysteroids) have
been identified in plants, with 20-hydroxyecdysone (20E) being the most
common one reported (Horn and Bergamasco 1985; Lafont and Horn
1989). The biological activity of some phytoecdysteroids has been tested by
a variety of insect bioassays, with many of these structures demonstrating molting hormone activity (Bergamasco and Horn 1980; Kubo and
Klocke 1983; Mele et aL 1992; Slama et aL 1993). In addition, it has been
shown that ecdysteroids exhibit interesting pharmacological effects on mammals, including the stimulation of protein synthesis and the reduction of blood
glucose and cholesterol levels (Camps 1991, and references cited therein).
These compounds have been found in various plant structures, and recent
studies have demonstrated that active biosynthesis of phytoecdysteroids takes
place in young developing tissues and they are then transported to older ones
(Grebenok and Adler 1991; Tomas et aL 1993; Grebenok et aL 1994). The
biosynthesis and distribution of phytoecdysteroids have been recently re-

Department of Plant Genetics, IRTA, Centre de Cabrils, Ctra. de Cabrils sin, 08348 Cabrils,
Spain
2 Department of Biological Organic Chemistry, CID (CSIC), J. Girona, 18, 08034 Barcelona,
Spain
1

Biotechnology in Agriculture and Forestry, Vol. 41


Medicinal and Aromatic Plants X (ed. by y.P.S. Bajaj)
Springer-Verlag Berlin Heidelberg 1998

334

J. Messeguer et al.

Fig. 1. Plant of Polypodium vulgare growing under greenhouse conditions, showing sori

viewed by Camps (1991), Macek and Vanek (1994), and Adler and Grebenok
(1995).
The phytoecdysteroid content of P. vulgare has been previously reported
by several authors (Heinrich and Hoffmeister 1967, 1968; Jizba and Herout
1967; Jizba et al. 1967a,b; Herout 1970). The most abundant ecdysteroid
isolated from rhizomes is 20-hydroxyecdysone (20E) but minor amounts of
other phytoecdysteroids such as polypodine B (5,20E) and the parent
ecdysone (E) have also been found. Nevertheless, the contents reported are
variable, and, apparently, among other factors, one should consider the possible influence of microclimate due to geographic location as well as the season
of collection. Also, the part of the plant examined and, very likely, the age of
the field-collected material could give rise to such differences.
In a preliminary study carried out in our laboratory, the rhizome content
in 20E ranged from 0.4% of the dry weight for plants collected in January to
0.04% for those collected in October. These results prompted us to consider
the possible application of in vitro culture for mass production of 20E, in which
the seasonal and other variation factors could be avoided.

Polypodium vulgare L. (Wood Fern)

335

HO
HO

Ecdysone (E)
20-Hydroxyecdysone (20E)
Polypodine B (5,20E)
Abutasterone (20,24E)
5-Hydroxyabutasterone (5,20,24E)
24-Hydroxyecdysone (24E)
PteroSIerone (25d20,24E)
Inokosterone (25d20,26E)

R}

R2

R3

R4

Rs

H
H
OH
H
OH
H
H
H

H
OH
OH
OH
OH
H
OH
OH

H
H
H
OH
OH
OH
OH
H

OH
OH
OH
OH
OH
OH
H
H

H
H
H
H
H
H
H
OH

Fig. 2. Chemical structures of phytoecdysteroids isolated from P. vulgare. Abbreviations of


ecdysteroids based on Lafont et al. (1993)

2 In Vitro Culture Studies


2.1 Prothallus Production

Rhizomes were collected in the field and cultivated under greenhouse conditions until plants were well established. Mature spores were placed inside filter
paper bags and surface disinfected (5% aq. NaCIO containing a few drops of
Tween-20) for lOmin, followed by washing with sterile distilled water (3 X
lOmin). Using a sterile paintbrush, a few spores were placed on the surface of
half-strength MS culture medium (Murashige and Skoog 1962) supplemented
with sucrose (30g/l) and Bacto Difco agar (8g/l), pH adjusted to 5.7 before
autoclaving at 1 atm for 20min. After 8-10 weeks the small prothallus clusters
(Fig. 3) were transferred into 500-ml culture vessels containing 60ml of the
same culture medium. The most vigorous clusters were selected and prothallus
lines were established by systematically subculturing them each 6 weeks, dividing into three to four sections. All cultures were incubated at 25 2 DC under
16-h light (Sylvania cool white, 37,uM/sm2 ).
After some subcultures, the growth pattern of prothallus-selected lines
was analyzed. As is shown in Fig. 4, during the first 6 weeks there was a parallel
increase between fresh and dry weight of prothallus clusters; then the dry
weight increased very slowly, whereas the fresh weight maintained its rate of
increase; after 60 days, both rates had almost leveled off and finally the fresh
weight even showed a small decrease, which was rationalized as the early

1. Messeguer et al.

336

Fig. 3. Prothalli of P. vulgare after 6 weeks in culture on MS basal medium

400

r----------------,

40

at

200

20

-<

-& fr. wt

"'dry wt
0'----------------10
o
~
00
1~
Time (days)

Fig. 4. Time course of P. vulgare prothalli growth. (Camps et al. 1990a)

Polypodiurn vulgare L. (Wood Fern)

337

symptom of a slight tissue necrosis, probably due to lack of nutrients (Camps


et al. 1990a).
2.2 Sporophyte and Callus Culture

The obtaining of sporophyte cultures from in vitro introduction of rhizome


segments was not possible, due to high infection of the plant material. Nevertheless, when prothallus clusters were cultured in the same medium, but
the agar concentration increased up to 12g/l, some sporophytes could be
isolated. This material was transferred into fresh medium and systematically
subcultured every 8 weeks. Cultures were incubated at 25 2C under 16-h
light (Sylvania cool white, 37.uM/sm2).
Callus was induced in prothallus clusters cultured in MS basal medium
supplemented with 3 mg/I 2,4 D. After a few days in culture, an increase in
prothallus thickness was observed. The formation of undifferentiated cell
clusters in the upper border of the prothalli was clearly distinguishable after 4
weeks. A green friable callus could then be isolated from individual prothalli
and subcultured in Petri dishes, in the same culture medium (Fig. 5). All
cultures were incubated at 25 2C under 16-h light (Sylvania cool white,
5 .uM/s m2). Selected callus lines could be maintained under in vitro conditions
by subculturing each 6 weeks.

Fig. 5. Callus cultures obtained from P. vulgare pro thalli, after 6 weeks in culture on MS basal
medium supplemented with 2 mg/l 2,4-0

338

1. Messeguer et al.

3 Phytoecdysteroid Content in In Vitro Cultures


Initial analysis of ecdysteroid content in in vitro prothallus cultures carried
out in our laboratory demonstrated that phytoecdysteroid production
increased continuously from the 1st week until 6 weeks in culture (Camps
et al. 1990a). Moreover, these prothalli showed a higher amount of 20E
(0.7% dry weight) and a lower one of polypodine B (5,20E) (0.1%) and E
(0.06%) when compared with those already found in field-collected or
greenhouse-cultivated plants. These results were of interest, because the
similar polarity of 20E and 5,20E renders their separation difficult. Thus,
reduced levels of polypodine B would greatly facilitate the purification
of 20E.
The prothallus cultures were also used to improve extraction and
phytoecdysteroid identification methods such as high-performance liquid
chromatography with UV (Camps et al. 1990b; Morgan and Marco 1990;
Coll et al. 1994) or mass spectrometry detection (Marco et al. 1993). Using
the improved technique (described in the extraction protocol), we identified
five phytoecdysteroids not previously reported in P. vulgare (Coll et al.
1994). Among them, inokosterone, pterosterone, and abutasterone had
already been identified in other plants, whereas 24-hydroxyecdysone and 5hydroxyabutasterone were described for the first time (Fig. 2).
More recently, Reixach et al. (1996) have analyzed the ecdysteroid content of different parts of wild P. vulgare plants collected in Spain and compared it with ecdysteroid content of in vitro-cultured tissues derived from the
same source. As shown in Table 1, all ecdysteroids found in the wild plant
are present in the in vitro-cultured tissues and vice versa. Rhizomes exhibited
the highest total production of ecdysteroids in the wild plant. On the other
hand, the methanol extracts of spores obtained from the same fronds as those
from which prothalli were obtained, did not contain ecdysteroids within
the detection limit of the analytical method used (10 f,lg ecdysteroid/g dry
weight).
The in vitro-cultured sporophytes present a morphological shape
and ecdysteroid content similar to those of the wild plant. Regarding the
Table 1. Ecdysteroid content of different parts of P. vulgare cultivated under in vitro and in vivo
conditions (mg ecdysteroid/g dry wt, mean SD, n = 4). (Reixach et al. 1996)
Rhizomes
20,24E
5,20E
20E
25d20,26E
24E
25d20,24E
E
a

0.02
2.70
4.23
0.06
0.09
0.03
0.03

Below detection limit.

Fronds

Sporophyte
in vitro

0.08 0.021
1.15 0.347

1.03
1.81
0.71
0.08
0.07
0.51

0.04

0.65
0.58
0.03
0.05
0.01
0.05

0.14 0.029

0.17
0.14
0.08
0.02
0.06
0.11

Prothalli
in vitro
0.34
1.00
5.98
1.07
0.19
0.29
0.67

0.02
0.05
0.26
0.03
0.01
0.01
0.03

Polypodium vulgare L. (Wood Fern)

339

most abundant ecdysteroids in the wild plant, the 20E: 5,20E ratio in
the in vitro-cultured sporophyte (1.76) is closely related to that obtained
considering together all parts of the wild sporophyte (1.86). This fact suggests
that the biosynthesis of ecdysteroids in the in vitro-cultured sporophyte
could be comparable to that of the wild plant in terms of balance of
compounds and total activity (4.42 vs. 4.77 mg ecdysteroids/g dry weight,
respectively).
Establised callus lines derived from prothallus cultures did not produce
any phytoecdysteroid. Nevertheless, during the process of callus formation,
prothallus samples were analyzed every 3 days and it was found that, as
pro thalli increase their thickness and begin to produce callus cell clusters,
the phytoecdysteroid content strongly decreased (data not shown). Thus, the
biosynthetic ability for secondary metabolites has been lost during the
dedifferentiation process as reported for many other species (Banthorpe
1994).
As had been previously described by Camps et al. (1990a), the
highest production of ecdysteroids was achieved in the in vitromicropropagated prothalli. The ecdysteroid profile of prothalli could be
considered similar to that obtained from wild fronds: both tissues produce
the highest proportion of 20E in comparison with the contents of the
other ecdysteroids. The relative amounts of E and especially 25d20,26E, in
both in vitro sporophyte and prothallus cultures, are remarkably high when
compared with the corresponding contents in the wild plant (Reixach et al.
1996).
Prothalli derived from spores collected in different European locations
[Ahrensburg (Germany), Espoo (Finland) and Montseny (Spain)] have been
micropropagated in vitro for 6-8 years. The fact that the growth ratio and the
shape of the prothallus cultures had been maintained for more than 60 subcultures indicated that these cultures could be considered morphologically stable
for all three sources tested. The total ecdysteroid production of prothalli
from different origins is summarized in Table 2 (Reixach et al. 1996). The
phytoecdysteroid production also appeared to be stable, since it was maintained at high levels during the years analyzed. The observed differences

Table 2. Total ecdysteroid content in prothalli derived from spores of P. vulgare from different
geographical origin micropropagated in vitro for 4 consecutive years. Data are given in mg
ecdysteroid/g dry wt (mean, n = 6). (Reixach et al. 1996)

1990
1991
1992
1993
Average

Ahrensburg
(Germany)

Espoo
(Finland)

Montseny
(Spain)

Average

5.85
8.01
6.45
6.83
6.84b"

9.76
10.39
8.99
10.58
9.93c

9.14
9.40
9.55
9.20
9.44c

8.25a
9.27a
8.33a
9.11a

, Statistical significance among means is indicated by different letters (ANOVA followed by


Tuckey's LSD test P = 0.01).

1. Messeguer et al.

340

Ahrensburg
6

Montseny

Espoo

10

3 4

II

;;;;;;~ 2

III

20E

25d20,24E ~ 24E

[3 5,20E

20,24E

25d20,26E

Fig. 6. Relative content of ecdysteroids in cultured prothalli derived from spores of P. vulgare
from different geographical origin. (Reixach et al. 1996)

were not significant and were probably due to variations in the subculturing
process, which was established for prothallus production every 6 weeks,
approximately. Nevertheless, the ANOV A analysis of these data detected
significant differences depending on the origin; thus, the phytoecdysteroid
productions of pro thalli derived from Espoo or Montseny spores were
similar, whereas pro thalli derived from spores from Ahrensburg were less
productive.
The separate phytoecdysteroid content for 4 consecutive years in P.
vulgare prothalli derived from spores collected in the above-mentioned
areas was also analyzed. No significant differences for each ecdysteroid production were detected among the different years analyzed (Reixach et al.
1996). In relation to the produced phytoecdysteroids (Fig. 6), although
20E is the major component, ranging from 66-70% for all the sources
studied, the percentage of the other phytoecdysteroids is distinctive for
each origin and can be used as a fingerprint character. Thus, ecdysteroid
production in prothalli generated from Ahrensburg spores shows a higher
proportion of E and 24-hydroxylated derivatives (25d20,24E; 24E; and
20,24E) and lower contents of 5,20E and 25d20,26E. Conversely, prothalli
derived from Montseny spores present an inverted profile related to the
former with higher proportion of 5,20E and 25d20,26E and lower one for
24-hydroxylated derivatives. Finally, ecdysteroid production in pro thalli
generated from Espoo spores exhibits a higher abundance of polar
ecdysteroids (20,24E and 5,20E) as well as E, and very low amounts of all
other compounds.
These differences in the biosynthetical pattern of in vitro prothallus culture from different sources and the fact that pro thalli have a simple morphology with natural laminar growth pattern, prompted us to use these cultures for
biosynthetic studies.

Polypodium vulgare L. (Wood Fern)

341

4 Biosynthetic Studies of Ecdysteroids in In Vitro Cultures


P. vulgare was one of the first plants used for ecdysteroid biosynthesis
studies. In 1970, de Souza et aI., using whole plant, found that [2_14C]_
mevalonic acid was converted to 20E, whereas [4- 14C]-cholesterol was
biotransformed to E, 20E, and 5,20E. The same plant material was used by
Davies et ai. (1980) to study the mechanism of formation of the A-B cis ring
junction of ecdysteroids.
More recently, Adler and Grebenok (1995, and references cited therein),
using Spinacea oleacea, have made an important contribution to better understanding of the regulatory role of ecdysteroid conjugates. Phosphates and
polyphosphate-ecdysteroids exert an inhibitory function on the biosynthesis of
ecdysteroids in this plant. A similar regulatory mechanism has been postulated
by Devarenne et ai. (1995) in Zea mays. On the other hand, in vitro cultures of
hairy roots of Ajuga replans have been used by Nagakari et ai. (1994a,b) to
show that [2- 13 C]-acetate and 3,B-hydroxy-5,B-cholest-7 -en-6-one are involved
in the 20-hydroxyecdysone biosynthesis.
4.1 Biotransformation of Putative Precursors of Ecdysteroids in Prothalli

The particularly simple morphology of prothalli (see Sect. 2.1, and Fig. 3) and
its natural laminar growth allows easy access to enzymatic systems for the
biosynthetic precursors artificially supplied. Putative precursors could be topically applied with a microsyringe in water or water: acetone solution.
From the biosynthetic point of view, ecdysteroids present in P. vulgare can
be separated in two main groups: 25-hydroxyecdysteroids (E; 20E; 24E;
20,24E, and 5,20E) and 25-deoxyecdysteroids (25d20,24E and 25d20,26E; see
Fig. 2).
In order to study the relationship between 25-hydroxyecdysteroids and its
plausible immediate precursor E, [23,24-'H]-ecdysone was applied to P.
vulgare prothalli derived from spores collected in Montseny and Ahrensburg
(Table 3; Reixach et ai. 1996). After 1 week, prothalli derived from Montseny
spores metabolized a 38% of [23,24- 3H]-ecdysone to ['H]-20E, and a small
amount (3 %) of [3 H ]-5,20E was also formed. The biotransformation of
the precursor increased up to 61 % after 3 weeks and, at that moment,
radiolabeled 20,24E (3%) was also present. The fact that [3H]-24E had not
been detected may suggest that 20,24E is biosynthesized, at least in part, via a
24-hydroxylation of 20E and not by the 20-hydroxylation of 24E. For elucidating the biogenesis of24E, prothalli derived from Ahrensburg spores, which are
a good source of 24-hydroxylated ecdysteroids (see Fig. 6), were treated with
[23,24}H]-ecdysone. As shown in Table 3, after 3 weeks, all detected 25hydroxyecdysteroids, including 24E (2%), showed 3H incorporation. Nevertheless, the relative amount of labeled 20,24E with respect to control (2: 10)
was the lowest when compared to the relative ratios of the other labeled
ecdysteroids (2: 6 as minimum). This fact suggests that ecdysteroids other than

342

J. Messeguer et al.

Table 3. Percentage of radiolabeled 25-hydroxyecdysteroids formed after incubation of P. vulgare

prothalli in the presence of [23,24-3H)-ecdysone. (Reixach et al. 1996)


20,24E
Montseny
Control"
1 week
2 weeks
3 weeks
Ahrensburg
Control a
3 weeks

5,20E

20E

24E

76
38
48
54

8
3
4
4

7
59
48
39

11
2

3
1

69
55

6
2

11
40

Percentages of 25-hydroxyecdysteroids produced in assays carried out in absence of labeled


precursors. These percentages are conserved through the 3-week period.

24E and 20E may be the precursor(s) for 20,24E. On the other hand, as
expected, the 25-deoxyecdysteroids were not labeled when [23,24)H]ecdysone was used as precursor.
As a means to study the biogenesis of all ecdysteroids present in P.
vulgare, an early biosynthetic radiolabeled precursor as [2- 14 C]mevalonic acid
(applied as a salt) was incorporated to in vitro-cultured prothalli (Reixach et
al. 1996). As shown in Fig. 7A, all identified phytoecdysteroids showed 14C
incorporation after 1 week in culture. Concerning the formation of 25deoxyecdysteroids, C4C]-25d20,26E was formed at the same rate as the
unlabeled one. Conversely, [14C]-25d20,24E was present in a higher amount
than in the control after the 1st week, but its contents decreased after 3 weeks
to become similar to those of the unlabeled analog. On the other hand, a
possible precursor of 25-deoxyecdysteroids (ponasterone A, 25d20E) was
tentatively identified in very low amount (1 %) in these assays. The fact that
this phytoecdysteroid has not been detected before in prothallus cultures
may suggest that it undergoes a rapid conversion. With respect to 25hydroxyecdysteroids, P4C]-E was present in high amounts during all the assay,
and although its relative contents decreased when the culture periods were
prolonged, the value after the 3-week period did not reach that of the control.
This relative decrease was associated with a relative increase of labeled polar
ecdysteroids, predominantly [14C]-20E.
When [4- 14 C]-cholesterol was fed as precursor, incorporation of radioactivity to all reported ecdysteroids was also observed (Fig. 7B; Reixach
et al. 1996). The relative incorporation profiles were similar to those obtained
with mevalonate, but with a lower proportion of E. This fact may indicate
that, although cholesterol is transformed into ecdysteroids, it may follow a
different biosynthetic pathway from that of the C27-sterol produced from
mevalonate, which should be the authentic biosynthetic precursor. In this
respect, Grebenok and Adler (1993) reported that although cholesterol
may be incorporated to ecdysteroids in Spinacia oleracea, lathosterol appeared to be the endogenous biosynthetic precursor of ecdysteroids in this
plant.

Polypodium vulgare L. (Wood Fern)

343

CONTROL
6

0
0..

20E

II

5,20E

lSI

24E

E:J

20.24E
25d20.24E
25d20.26E

1 week

2 week

3 week

Fig.7A,B. Percentage of radiolabeled ecdysteroids after incubation of P. vulgare prothalli in the


presence of A [2-'4C]-mevalonate and B [4-'4C] -cholesterol. Control Without radiolabeled precursors. (Reixach et al. 1996)

4.2 Biotransformation of Putative Precursors of Ecdysteroids in Calli

As previously mentioned (Sect. 2.3), callus derived from prothallus culture


(with a high ecdysteroid content) had lost its ability to produce these secondary metabolites. This fact makes calli a good model to check which
biosynthetic step(s) is( are) missing in this undifferentiated tissue and to contribute to better understanding phytoecdysteroid biosynthesis.
Putative biosynthetic precursors, such as [2- 14C]-mevalonate or [4_14C]_
cholesterol, that had been effectively biotransformed to ecdysteroids by
prothalli, were applied to P. vulgare calli. Although these experiments were

1. Messeguer et a!.

344

100

(:I;l

'i

75

oS.,

=
f

50

:E
~

25

Time (days)
Fig. 8. Time course biotransformation of E in P. vulgare callus culture. (Unpub!')

carried out up to 5 weeks, incorporation of radioactivity to the ecdysteroid


fraction was not observed.
On the contrary, when [23,24- 3H]-ecdysone was applied to P. vulgare calli,
it was efficiently biotransformed (Fig. 8; unpubl.). However, some differences
between the oxidative capacity of pro thalli and calli were observed. First
of all, although from the radioactive point of view it seems that the
biotransformation is faster in calli than in prothalli (75% after 1 week in calli
versus 40% in prothalli), it should be kept in mind that radiolabeled ecdysone
is diluted into the endogenous one in pro thalli. With this consideration,
prothalli are actually more active than calli. On the other hand, only 20E
was observed all through the time in calli, even when 90% of labeled ecdysone
was biotransformed, whereas prothalli produced all radiolabeled 25hydroxyecdysteroids when [3 H ]-ecdysone was applied (Table 3).

5 Summary and Conclusion


Prothallus clusters of Polypodium vulgare cultured in vitro are an excellent
material for studying the content and the biosynthetic pattern of
phytoecdysteroids. These cultures could be maintained in vitro for a long time
in such a way that variations related to climatic conditions or due to the
physiological stage of the plant could be avoided. On the other hand, their
phytoecdysteroid production is constant and reproducible for all sources
tested. Moreover, their natural laminar growth allows an easy incorporation of
biosynthetic precursors and/or modulators artificially supplied.
Labeled mevalonate, cholesterol, or ecdysone can be incorporated and
biotransformed to ecdysteroids by in vitro-cultured prothalli. As shown
in Fig. 9, labeled ecdysone may partially be a biosynthetic precursor for

Polypodium vulgare L. (Wood Fern)

345

___ 20E

MVA - - - - - Cholesterol _____

E--...,. 24E

5,20E

~O,24E

",/ 25d20,24E /
25d20E

"'" 25d20,26E

Fig.9. Last steps of biosynthetic pathway of phytoecdysteroids in P. vulgare prothalli. (Reixach et


al. 1996)

25-hydroxyecdysteroids, whereas mevalonate and cholesterol may be


biotransformed to all, up to now, known ecdysteroids including those recently
identified. [3H]-Ecdysone was biotransformed to 20E and 24E, but no
matter what labeled precursor had been used, the ratio of labeled E: 20E (or
E: 24E) was always higher than that of the control. The ratio of labeled
20E: 5,20E in cultures carried out with [3H]-ecdysone and [14C]-mevalonate
was close to that of the control (Table 3, Fig. 7A), which indicates that 20E
is the main biosynthetic precursor of 5,20E. On the other hand, when
[3H]-ecdysone was used as precursor, labeled 20,24E was observed only after
3 weeks (Table 3), whereas in experiments that allowed the formation of
labeled 25d20,24E (Fig. 7), the percentage of this ecdysteroid decreased with
time concomitantly with an increase in labeled 20,24E. These results may
indicate that 20E, 24E, and 25d20,24E contribute to the biosynthesis of
20,24E.
Despite the fact that callus cultures have lost their ability to produce
phytoecdysteroids, they are able to biosynthesize 20E from E, which
makes these materials also very useful for studying phytoecdysteroids
biosynthesis.
The distinct behavior between these two in vitro-cultured materials in
respect to the incorporation of different precursors could help to elucidate the
biosynthetic steps and clarify which are the enzymatic systems that have been
lost during the dedifferentiation process of callus cultures.
To conclude, prothallus and callus cultures of P. vulgare are excellent
tools to carry out biosynthetic studies of phytoecdysteroids.

6 Protocol for Extraction and Quantification of


Ecdysteroids in P. vulgare
Chromatographic procedures for phytoecdysteroid purification and isolation have been recently
reviewed (Lafont et al. 1994a; Macek and Vanek 1994). Different approaches, depending on the
plant source (roots, leaves, etc.) and amount (analytical or preparative scale) are suggested.
Briefly, the analysis of ecdysteroids in analytical scale involves an extraction with methanol and a
purification with disposable cartridges.
P. vulgare phytoecdysteroid contents discussed in Section 2 were obtained with the following
procedure. Lyophilized pro thalli or sporophytes (micropropagated in vitro), rhizomes, or fronds

346

1. Messeguer et al.

(50 mg) were extracted with MeOH (4 X 5 ml) and the combined extracts were evaporated. The
residue was dissolved in 2 X 5 ml of H,O and loaded onto a CI8 reversed-phase Sep-Pak cartridge.
After washing with H 20 (lOml) and 15:85 MeOH:H,O (lOml), ecdysteroids were eluted
with 85: 15 MeOH: H 20 (5 ml). An aliquot of this eluate was mixed with 7-ethoxycoumarin
(used as internal standard for quantification) and injected onto the HPLC system (Reixach et al.
1996).

On the other hand, when biosynthetic studies of phytoecdysteroids using


radiolabeled precursors (see Sect. 3) were carried out, a smaller proportion of
tissue was used (approx. 20-30mg fresh weight). In order to avoid radioactivity manipulation as much as possible, a simple analytical procedure was developed. Samples were extracted with water (2ml) at 80C for 1h. With this
procedure, less than 20% of ecdysteroids remained in the tissue. The water
extract obtained may be injected into the HPLC system without further
manipulation.
As already mentioned above, a large number of chromatographic conditions for HPLC separation of ecdysteroids have been published and most of
them have been included in a recent review by Lafont et al. (1994b). However,
due to the high number of ecdysteroids present in P. vulgare with a very
closely related chemical structure, new HPLC conditions for a baseline separation of ecdysteroids in reversed phase were established (ColI et al. 1994).
The reversed-phase HPLC column was a Spherisorb ODS2 (5.um, 15cm X
4.6mm i.d.) maintained at 55C and eluted with isopropanol:water (7:93)
for 10min followed by a linear gradient (over lOmin) from 0 to 20% of
acetonitrile in isopropanol: water (7: 93) at 1.2mllmin. Analyses were monitored at 242nm with a diode array detector and on-line HPLC radioactivity
monitor (when needed) and recorded and quantified with a chromatographic
software.
Acknowledgments. The authors thank Dr. F. Camps for critical review of the manuscript. Financial support from CICYT (Grant BIO/92/0398 and PB/94-0083) and predoctoral fellowships from
CIRlT-Generalitat de Catalunya (to N.R.) and the Ministerio de Educaci6n y Ciencia (to J.l.-S.)
are acknowledged.

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Bergamasco R, Horn DHS (1980) The biological activity of ecdysteroids and ecdysteroid
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XVIII Rosmarinus officinalis L. (Rosemary): In


Vitro Culture, Regeneration of Plants, and the Level
of Essential Oil and Monoterpenoid Constituents

1 General Account
1.1 Importance of Rosemary

Rosemary (Rosmarinus officinalis L., family Labiatae), a plant native to Mediterranean countries, is an evergreen shrub (Fig. 1) commonly grown for extraction of its aromatic oils by steam distillation. About 100 metric tons of its oil are
produced each year, mainly in Spain, Italy, France, Yugoslavia, the former
USSR, Tunisia, Morocco, and the Middle East (Browse 1986). Spain is the
largest producer of rosemary. Several species of the genus Rosmarinus with
a number of varieties are commonly known (Guenther 1949). The oil yield
is much higher during summer than in winter. In summer, 12 arrobas
(1 arroba = 11.5 kg or about 25Ib.) of plant materials yield about one kg of oil,
while in winter 20 arrobas are required. The properties of rosemary oil vary
according to several environmental factors associated with the producing region, e.g., soil, climate, and season of harvesting (Guenther 1949). Sovoboda
and Deans (1992) reported that the anti oxidative properties of various samples
collected from different dealers and markets varied according to geographical
location and type of processing. The aromatic oil is used widely in the perfume
industry. Rosemary is also used in food processing as a preservative. Its
essential oils have been shown to inhibit several Gram-positive and -negative
bacteria (Narasimha Rao and Nigam 1970). Generally, Gram-positive bacteria
are more sensitive to rosemary and sage oils than Gram-negative ones. A
relationship between the chemical structures of the most abundant components in the essential oils and the antimicrobial activity was found (Farag et al.
1989). Shelef et al. (1980) reported that the inhibitory effect of rosemary and
sage was attributed to their terpene fraction, which was comprised of borneole,
cineole, pinene, camphene, and camphor for rosemary, and thujone for sage.
The antioxidative activity of rosemary has been shown by other authors.
Nakatani and Inatani (1984) isolated two diterpenes that showed four times
higher antioxidative activity in lard than synthetic antioxidants such as
Horticulture Department, College of Agriculture, Assiut University. Assiut 71526 Egypt
Horticulture Department, University of Nebraska, Lincoln, Nebraska 168583-0724,
USA
3 Department of Food Sciences and Technology, University of Nebraska, Lincoln Nebraska
168583-0919, USA
1

Biotechnology in Agriculture and Forestry, Vol. 41


Medicinal and Aromatic Plants X (ed. by y.P.S. Bajaj)
Springer-Verlag Berlin Heidelberg 199H

350

A.A. Tawfik et al.

Fig.i. Two cultivars of rosemary, Rosmarinus officinalis L., used in this study: left Prostratus; right
Lockwood de Forest

butylated hydroxy anisole (BHA) and butylated hydroxy toluene (BHT).


Banthorpe et al. (1989) reported that the (Z,E) and (E,E) isomers of the enol
ester were found in the foliage and cell cultures of seven out of eight species of
the family Labiatae screened (e.g., Rosmarinus officinalis and Salvia
officinalis). In a test with Cladosporium herbarium, these pigments exhibited
some fungicidal activities. Research at the University of Nebraska, focused on
identifying compounds in rosemary that have antioxidant properties, showed
that rosmariquinone was effective in inhibiting both photo-and auto oxidation
in soybean oil (Cuppett et al. 1992). Rosemary is also classified as a medicinal
plant, the leaves being traditionally used as a stomachic stimulant and hepatic
agent. The extract of the dried leaves was found to exhibit marked inhibitory
activities against KB cells (87%) at 50,ug/ml (Hayashi et al. 1987). The KB
assay is a well-known method to screen anticancer agents in natural resources.
In a study by Cuppett et al. (1995), rosmariquinone, a diterpene extracted from
rosemary leaves, was found to exhibit a potent antimutagenic activity in V79
cells treated with methyl-N-nitrosourea (MNU). Some studies have provided
evidence for an increasing diversity of naturally occurring compounds having
the capacity to inhibit nitrosamine carcinogenesis. Wattenberg et al. (1989)
reported that limonene (Figs. 2, 3), a constituent of rosemary oil, reduced the
chances of forestomach tumor formation by more than 60%. Camphor is
another component of rosemary oil and has been reported to stimulate the
activity of the central nervous system, respiration, and circulation. Hoefler et
al. (1987) reported that the ethanol extracts of young shoots of rosemary
markedly stimulated bile secretion. Aqueous extracts of young shoots showed
significant hepatoprotective properties when given to laboratory animals

Rosmarinus officinalis L. (Rosemary)

351

as a pretreatment before carbon tetrachloride administration. These results


support the traditional medicinal uses of this plant in the treatment of liver
disorder. The aqueous extract of rosemary is used to treat abdominal colic.
AQel (1991) found that the essential oil of rosemary leaf inhibited muscle
contractions induced by acetylcholine, histamine, and a solution containing K+
at 60mM. The oil also inhibited muscle contractions induced by acetylcholine
or histamine in Ca2 + -free solution. The results suggest that the oil may have Caantagonistic properties. In an attempt to identify the active components of
Rosmarinus officinalis and Gentiana lutea, used in the treatment of hepaticbiliary deficiency and to help digestion, Verotta (1985) described the isolation
of rosmarinic acid from the two species.
1.2 Composition of Rosemary Oil

The rosemary flavor is due to a mixture of compounds in its essential oil. The
oil contains mainly monoterpenes, some diterpenes, and sesquiterpenes. The
group of structurally related 10 carbon compounds, called monoterpenes, is
usually constituted of lower boiling fractions of essential oils (Croteau 1980).
Another group of volatile compounds called sesquiterpenes, with higher boiling fractions and 15 carbon atoms, contributes to the odor and flavor of
essential oils. Higher terpenes such as diterpenes (C-20) are readily eliminated
by distillation. Sesquiterpenoids are often found as components of volatile oils
extracted by steam distillation. Essential oils of three species of rosemary (R.
officinalis, R. eriocalyx, and R. tomentosus) were analyzed by gas chromatography (GC) and mass spectrometery (MS) (Rosua and Gareia-Grandos 1987).
These authors found 15 components (13 monoterpenes and 2 sesquiterpenes)
and the most abundant components of oil from the three species were apinene, camphor, 1,8-cineole and a-terpineol. Tucker and Maciarello (1986)
reported that 23 cultivars of the rosemary, Rosmarinus officinalis, representing
five botanical varieties, showed a wide range in the leading components:
0.6-57.45% a-pinene, 3.55-42.69% 1,8-cineole, 0.20-56.45% camphor, 0.6621.03% bornyl acetate, and 0.40-14.69% borneol. Ten monoterpenes have
been identified in the oil of both rosemary's genotypes R. officinalis L.
Prostratus and R.o. Lockwood de Forest using GC/MS (Tawfik 1992): apinene, j3-pinene, camphene, p-cymene, 1,8-cineole, limonene, linalool, camphor, borneol, and bornyl acetate (Fig. 3). The chemical structure of these
monoterpenes is shown in Fig. 2.
1.3 Extraction and Separation of Rosemary Oil

The basic method that is commercially used for obtaining the aromatic oils
from rosemary plants is steam distillation. However, many techniques have
been employed for essential oil extraction, such as organic solvent extraction.
The extraction method of choice depends on the nature of the compound of
interest and the amount of plant material used for extraction. The oil is a
colorless or pale yellow liquid having the characteristic odor of rosemary and
a camphoraceous taste.

352

A.A. Tawfik et al.

pinene <alpha>

6J
ex 2

Camphor

1,S-clneole

pinene <beta>

Camphene

Borneol

Limonene

OH

Y'

o
I

C}l"OCCH,

~
Bornyl acetate
p-cymene

Llnalool

Fig. 2. Chemical structure of monoterpenes identified in rosemary oils

For determining the levels of monoterpenes in proliferated explant and


regenerated plantlets, extraction using hexane as an organic solvent was considered to be the preferable method to use. The plant tissues were placed in
glass vials and thoroughly soaked in a sufficient amount of hexane (2 mllO.5 g
fresh wt.). The vials were then capped and sealed, and extraction continued for
12-15 h at 4C. Water was removed from the extract using sodium sulfate
anhydrous (Na 2S04 ) then decanted into clean vials, and the final volume for all
treatments was adjusted to 2ml using hexane. A 2-pl aliquot of the oil extract
was injected into a Hewlett Packard Gas Chromatograph (GC) Model 5890A
using a flame ionization detector (FID) temperature of 350C and an injection
temperature of200C. A splitless injection with the purge offfor 0.75 min was
made onto a 30-m SE-30 capillary column incrementally programmed from
60C (2-min hold) to 145C at 3C/min. FID output was electronically integrated and data reported as area percentages. Identification was done using

Rosmarinus officinalis L. (Rosemary)


12

353

I a-pinene
10

2 Camphene
3 Ll-pinene

4 p-cymene
5 I,S-cineole

6 Limonene
7 Linalool

8 Camphor
9 Borneol
10 Bornyl acetate
\

\
I

Fig. 3. Gas chromatogram of the oil of rosemary

the combination of standards and Hewlett Packard Gas Chromatograph-Mass


Spectrometer (Gc/MS Model 5870).

2 In Vitro Culture Studies


Cutting is the traditional method to propagate rosemary. Despite the importance of rosemary plants, research efforts to improve existing cultivars or to
produce new ones with higher quantity and quality of essential oil are limited.
Rosemary grows wild and is rarely cultivated except for ornamental purposes
(Guenther 1949). The variation in the physicochemical properties of its oil
may be caused by environmental conditions. Therefore, the improvement of
rosemary cultivars through in vitro culture is of great demand.
1. Callus Induction and Regeneration of Plants. The induction of organogenic
calli depends on growth regulators and the type of organ used, i.e., leaf
segments, stem segments, shoot tips, and/or meristem tips. For this research

A.A. Tawfik et al.

354

the foregoing explants were taken from two different genotypes of

Rosmarinus officinalis L. Prostratus and R.o. Lockwood de Forest (Fig. 1). All
explants taken from R.o. Lockwood de Forest induced dark green compact

calli when cultured on MS medium (Murashige and Skoog 1962) with the right
growth regulator. For the other genotype, Prostratus, shoot tips were the
better explant and induced excellent callus for plant regeneration on the same
medium.
A protocol to induce organogenic callus and to produce plantlets via
callus is employed (Tawfik 1992). Thidiazuron (TDZ) was better than 2,4dichlorophenoxyacetic acid (2,4-D) to induce organogenic calli. High concentrations of TDZ (up to 2mg/l) either alone or plus 0.5mg/l indole acetic acid
(IAA) induced organogenic callus.
2. Shoot- Tip Proliferation. In both the genotypes, the callus was formed on

the bases of the shoot tips cultured on MS medium supplemented with TDZ
alone or with IAA. The interaction of IAA by TDZ on the proliferated
explant fresh and dry weight was significant. The highest fresh and dry weight
of callus was obtained when 1.5 mg TDZ/I plus 0.5 mg IAA/I were applied to
the medium.
3. Stem Segment Proliferation. The stem segments of Lockwood de Forest

were excellent explants for producing large masses of organogenic callus when
cultured on the designated medium. No callus was formed on TDZ-free medium, and the presence of IAA in the callus induction medium (elM) was
essential to produce the largest mass of dark green organogenic callus. The

TDZ

0.0

0.5

1.5

mg/I

0.0
IAA

0.5
Callus from stem segment
Fig. 4. Callus proliferation from stem segments of Rosmarinus officinalis Lockwood de Forest 5
weeks after culture as affected by TDZ and IAA added to the culture medium

Rosmarinus officinalis L. (Rosemary)

355

highest fresh weight was obtained from stem segments cultured on medium
supplemented with 2mg TDZ/I plus 0.5mg IAA/I (Fig. 4).
4. Leaf Segment Proliferation. The same response of TDZ and IAA, when
applied to MS medium for stem segments cultured in vitro, was observed for
the leaf segments (Fig. 5). The callus was formed on the abaxial side of the leaf
segment. After 4-6 weeks, compact dark green organogenic calli were ready
to transfer to the regeneration medium.

TDZ (mg/l)

,.....

0.5

.,.

0.5

1.5

Callus from leaf segment


Fig. 5. Effect of TDZ and IAA on the fresh weight of Rosmarinus officinalis Lockwood de Forest
induced from leaf segments 5 weeks after culture in vitro

5. Plant Regeneration. When the compact dark green healthy callus from
both genotypes was transferred to a medium supplemented with different
concentrations of benzyl adenine (BA) (0, 2, 4, 6, and Smg BAil), multiple
shoots were produced (Fig. 6). The best concentrations were 4 and 6mg
BAIL

3 Effect of Medium Composition on Growth and


Monoterpene Levels
For all the following studies, the protocol mentioned above using either shoot
tip or leaf segments was used.

A.A. Tawfik et al.

356

Fig. 6. Shoot regeneration of rosemary Rosmarinus officinalis L. Prostratus from callus 6 weeks
after culture as affected by BA concentrations added to the culture medium

3.1 Effect of Sucrose

The growth responses to sucrose as a source of carbohydrate differed, depending on the genotype used. Increasing sucrose concentrations in the medium
resulted in an increase in fresh and dry weight of the proliferated explant
(shoot tips) taken from Prostratus (Table 1). However, a high concentration of
sucrose (40mg/l) resulted in decreasing the fresh and dry weight of the two
different explants (leaf segments and shoot tips) taken from R.o.L. Lockwood
de Forest (Table 2). Maretzki et a1. (1974) reported that the effect of carbohyTable 1. Effect of sucrose on the proliferated explants of
Rosmarinus officinalis Prostratus 5 weeks after culture in vitro

Sucrose
conc. (gil)

Fresh weight
(g)

Dry weight
(g)

Bornyl acetate
(%)'

10
20
30
40

0.11
0.30
0.68
1.11

0.030
0.047
0.066
0.084

3.7
4.3
7.5
7.2

Contrast b

L**

L**

L*

, % of the total mono terpene in the essential oil extracted from


the proliferated explants. To convert to flg/g fresh wt., multiply by
(5 X 10- 4 ).
b L = linear. Contrasts are significant at P < 0.05 (*) or P < 0.01
(**), respectively.

Rosmarinus officinalis L. (Rosemary)

357

Table 2. Effect of sucrose on the proliferated explants of Rosmarinus officinalis Lockwood de


Forest 5 weeks after culture in vitro

Sucrose
conc. (gil)

CFW'(g)

CDW (g)

STFW (g)

STDW (g)

j3-Pinene

10
20
30
40

0.99
1.26
1.60
0.90

0.036
0.080
0.092
0.050

0.38
0.49
0.37
0.15

0.035
0.048
0.053
0.026

6.1
7.3
6.0
3.4

6.9
7.6
6.0
4.0

Contrast'

Q*

Q**

Q**

Q**

Q**

L*

Borneol

(%)b

, CFW = callus fresh weight; CDW = callus dry weight; STFW = shoot tip fresh weight, STDW
= shoot tip dry weight.
b % of the total monoterpene in the essential oil extracted from the proliferated explants (shoot
tips). To convert to Ilglg fresh wt., multiply by (5 x 10- 4 ).
, L = linear, Q = quadratic. Contrasts are significant at P < 0.05 (*) or P < 0.01 (**), respectively.

drate on growth differed, depending on the species or clone, but not on the
tissue from which the explant was isolated.
The effect of sucrose concentrations applied to the culture medium on the
levels of the monoterpenes identified in the extract from the proliferated
explant of R.o.L. Prostratus was reported for bornyl acetate (Table 1). Increasing sucrose concentrations led to an increase in the level of bornyl acetate.
Calvo and Sanchez-Grass (1993) studied the accumulation of monoterpenes in
proliferated shoots of Lavandula latifolia Med, and found that increasing the
medium osmolarity by addition of mannitol to the culture medium resulted in
a significant increase in monoterpene accumulation in the regenerated shoots.
In R.o.L. Lockwood de Forest, the level of j3-pinene decreased when high
concentrations of sucrose were used (Table 2). Sucrose had a significant quadratic effect (P < 0.01) on the j3-pinene level. Based on the quadratic regression
line, the highest level of this monoterpene was estimated to be obtained when
approximately 199 sucrose!l was added to the culture medium (Neter and
Wasserman 1974). Norton et al. (1991) reported that rubber synthesis in
guayule (Parthenium argentarum), was stimulated by increasing the sucrose
concentration up to 7% in the medium, but higher concentrations were inhibitory. On the other hand, increasing sucrose concentration in the medium up to
40 g sucrose/l inhibited the borneol level in the proliferated explant of R.o.
Lockwood de Forest cultured in vitro in our studies (Table 2).
3.2 Effect of Calcium Ion

To study the effect of Ca2+ ion on callus induction and monoterpene levels of
Lockwood de Forest, leaf segments were used as explants and cultured on
callus induction medium (CIM) supplemented with six concentrations of Ca2+
ion (0.99, 1.99, 2.99, 3.99, 4.99, 5.99mMol!I). It was obvious that Ca 2+ ion
supplementation affected the texture of rosemary callus. The lowest concentration (0.99mMol Ca 2+/l) produced dark green and compact callus. The highest concentration (5.99mMol Ca2 + II) produced light green friable callus. The

A.A. Tawfik et al.

358
3

--~

J:
Cl

';

2.9
2.8
2.7

III
::l

2.6
2.5 0.99

1.99

2.99

3.99

4.99

5.99

ct + cone. mMol/L
0.17 r - - - - - - - - - - - - - - - - - - - - - ,

0.165 0.16 f- ........ ',............................... / ............................................\

................................................... .

1:

.~ 0.155
~

~ 0.15 .
III
::l

'iii
()

0.145
0.14
0.135

LL_ _ _"---_ _---'-_ _ _- ' -_ _ _' - -_ _-'...J

0.99

1.99

2.99

3.99

4.99

5.99

2+

Ca cone. mMol/L
Fig. 7A,B. Effect of Ca2 + concentration on the fresh weight (A) and the dry weight (B) of
Rosmarinus officinalis Lockwood de Forest callus proliferated in vitro

effect of calcium ion on fresh and dry weight is shown in Fig. 7A, B. The higher
concentration produced high fresh weight but low dry weight because of the
loss of moisture. However, no significant effect was found on both fresh and
dry weight. Murashige and Skoog (1962) observed that Ca2+ ion concentration
in the range of 1.5 to 6mMolli did not affect the fresh weight of tobacco pith
explants. Likewise, Gamborg et al. (1968) reported that Ca2 + ion concentrations from 1 to 4 mMolll did not affect the dry weight of soybean suspension
cultures. However, Mizukami et al. (1977) reported that increasing Ca2 + ion
concentration above 3 mMolll decreased growth in Lithospermum callus
cultures.
The calcium ion significantly affected the oil yield (P = 0.01) extracted
from the proliferated callus of rosemary. The oil yield curve, expressed
as a response to calcium ion concentrations, was cubic (Fig. 8). The highest yield of oil measured per g fresh weight was obtained from calli grown

Rosmarinus officinalis L. (Rosemary)

359

0.15

-~

0.14

Y = 0.34 - 0.27 X

0.13

R = 0.75

+ 0.086 X - 0.008 X

0.12

LL 0.11
1:1
0.1

#.

0.09
0.08
0.07
0.06
0.05

0.04
0.03

0.99

1.99

2.99

3.99

4.99

5.99

2+

Ca cone. mMoI/L

Fig. 8. Effect of calcium ion on oil yield extracted from proliferated callus of Rosmarinus
officinalis Lockwood de Forest

Table 3. Effect of calcium ion on oil costituents (%) extracted from Rosmarinus officinalis
Lockwood de Forest proliferated callus 6 weeks after culture in vitro

Ca2 + (mMol/l)

Oil constituents (%)'


Camphene

1,S-Cineole

Linalool

Bornyl acetate

0.99
1.99
2.99
3.99
4.99
5.99

6.S
3.4
3.1
3.0
5.5
5.2

20.9
24.5
24.0
27.0
21.5
lS.2

2.22
2.32
1.26
2.23
2.25
2.74

O.SO
0.75
1.45
O.SO
1.20
0.44

Contrast b

C**

Q**

Q*

C*

, % of the total mono terpene in the essential oil extracted from the proliferated callus. To convert
to flg/g fresh wt., multiply by (5 X 10- 4).
b Q = quadratic; C = cubic. Contrasts are significant at P < 0.05 (*) or P < 0.01 (**), respectively.

on a medium supplemented with the lowest concentration of Ca2 + ion


(O.99mMol/l).
Among the ten monoterpenes identified in the mother plant, only eight
were isolated and identified in the calli. Ca2+ ion affected only four of them:
camphene, 1,8-cineole, linalool, and bornyl acetate (Table 3). The calcium ion
quadratically influenced both 1,8-cineole and linalool. The maximal level of
1,8-cineole was found in callus induced on a medium supplemented with
approximately 3.2mMol Ca2+ 11. These results agreed with those reported by
Mizukami et al. (1977), who found that the yield of shikonin derivatives in
Lithospermum callus cultures decreased as the Ca 2+ ion concentration increased above 3 mMolll. Tawfik et al. (1992b), reported that the highest level

360

A.A. Tawfik et al.

of linalool was produced at S.99mMol Ca2 +!l. The lowest concentration of


calcium ion (0.99mMolll) produced the highest level of camphene.
3.3 Effect of Growth Regulators

Callus induction and plant regeneration of rosemary from all types of explants
using auxin and cytokinin have been described. For inducing organogenic
callus, a combination ofthidiazuron (TDZ) and 3-indoleacetic acid (IAA) was
applied to the culture medium. For plant regeneration, callus was transferred
to a medium supplemented with benzyladenine (BA).

3.3.1 Effect of TDZ and 1AA on Monoterpene Levels


For most of the monoterpenes identified in the proliferated explant of rosemary, a significant interaction of TDZ by IAA (P < 0.01) was observed (Fig.
9). The highest level of camphor (Fig. 9A) was obtained when O.Smg TDZ/I
plus 0.5 mg IAA/I were added to the culture medium. TDZ alone at 0.5 mg/l
produced the highest levels of 1,S-cineole (Fig. 9B) and borneol (Fig. 9C),
while the highest level of camphene (Fig. 9D) and bornyl acetate (Fig. 9E) was
obtained from a proliferated explant cultured on a medium supplemented
with 2mg TDZ/I alone. IAA added to the culture medium with TDZ seemed
to have an inhibitory effect on 1,S-cineole and borneol. However, the highest
level of limonene (Fig. 9F) was obtained when 0.5 mg IAA/I alone was used.
Both TDZ and IAA reduced the level of limonene extracted from proliferated
explant. The highest level of linalool (Fig. 9G) was obtained from proliferated
explant cultured on hormone-free medium.

3.3.2 Effect of BA on Monoterpene Levels


When the calli from shoot tips of Prostratus were cultured on 30ml of MS
medium supplemented with different concentrations of BA, all the treatments
led to shoot regeneration. The shoots from each treatment were transferred to
the greenhouse for rooting and further growth. When they reached 15 cm in
height, samples from each treatment were taken for oil extraction and analysis.
BA significantly influenced the levels of six of the monoterpenes identified: cx:_
pinene, j3-pinene, camphene, 1,S-cineole, camphor, and bornyl acetate (Table
4). The highest levels of cx:-pinene and camphene were found in plants regenerated on BA-free medium. However, BA at a high concentration (Smg/l)
increased the level of 1,S-cineole and camphor in the regenerant plants.
BA had a quadratic effect on j3-pinene and bornyl acetate (P = 0.1). Based
on the quadratic regression line, the highest levels of /3-pinene and bornyl
acetate were estimated to be in plants derived from medium supplemented
with approximately 3.7 and 3.9mg BAil, respectively (Neter and Wasserman
1974).

61

'b~><

9
8
7
8

(0)

(A)

A'"

~~

0.5

",

-0.0

TOZ mg/l

~0.5

1.5

2.0

"'.

mg/I

2.0

~0.5 m<>,

- _.'"

IAA---O.0

mg,'

1.0

TDZ

10
.

IAA

'.~~~
.--~
- --- ... - - - --~
-

23

::0-

51

0.0

~4

Q.

.c:

g 6'

III

~ I
-

lOj (0)

,i

or

54

0.5

.
2;0

~O

0.5 mg/I
1.5

IAA~O.O ~

1.0
TDZ mg/l

- '-

~- - -/-- /

1.0' "
1'5
TDZ m g / I '

"
,,,
'

..- -- - - - . -'- - - - - . -

05
.

A
.'

IAA ~~O.O ~. 0.5 mg/l

~~ i3l~ -------., .....

22
c: 21
.- 20

24

~ 25

~~

~~j

30j (8)

Fig.9A-G. Effect of TDZ and IAA on the mono terpene level (%) in rosemary shoot tips cultured in vitro. A
Camphor. B I.S-Cineole. C Borneol. D Camphene. E Bornyl acetate. F Limonene. G Linalool

~l~-------,

i
()

"5. 10

.12
5 11

-13

14

16
15

(.;.l

2i

:3-'"

(1)

C/O

~
o

(::)

<"l

'"
~
s

5"
;:

(::)

:;.;,
o

A.A. Tawfik et al.

362
7

!
QI

(E)

,,

,,

,,

>. 3
E

dl

"

1M

~ 0.0 ~O.5

mg/l

1.

1.0

2.0

TDZ mg/I

QI

c
QI
c

:::J

1.04 (F)
1.00 ' ,
,
0.96
,
,
0.92
0.88
0.84
0.80
0.76
0.72
0.68
0.64
0.60
0.56
0.0

1M n-+o().O

0.5

~ 0.5

.. "
,

,
,,

1.0

mg/I

"~
....
,

,,

,,

1.5

2.0

TDZ mg/I
4

(0)

'\

1M ....,..... 0.0 -0.5 mg/I

1.~~~~~~~~~~~~~~~

0.0

0.5

1.0

TDZ mg/l
Fig. 9E-G. Continued

1.5

2.0

Rosmarinus officinalis L. (Rosemary)

363

Table 4. Effect of BA on the oil constituents of Rosmarinus officinalis Prostratus plants regenerated from callus
BA (mg!l)

Oil constituents (% Y
a-Pinene

Camphene

{:i-Pinene

1,8-Cineole

Camphor

Bornyl acetate

0
2
4
6
8

8.0
8.2
8.1
7.0
7.1

4.4
4.5
4.2
3.6
3.4

4.5
4.7
5.1
4.8
4.2

23.5
24.1
23.2
26.0
26.1

18.0
16.9
16.0
16.3
18.7

6.7
7.3
8.1
7.7
6.4

Contrast"

L*

L*

Q*

L*

Q**

Q**

, % of the total mono terpene in the essential oil extracted from the regenerant plants. To convert
to JIg!g fresh wt., multiply by (5 X 10 4).
b L = linear, Q = quadratic. Contrasts are significant at P < 0.05 (*) or P < 0.01 (**), respectively.

It is clear that the production of monoterpene constituents from proliferated explants cultured in vitro and from regenerated plants was influenced by
the components of the culture media, such as plant growth regulators. These
results partially agree with the results reported for Mentha spicata (Hirata et
al. 1990) and Zingiber officinale (Sakamura et al. 1986). The effects of plant
growth regulators on the yield of some secondary products in plant cell cultures have been investigated by several groups (see Bajaj 1996). The effects
varied greatly, depending on the kinds of metabolites being produced and on
the type of auxin and cytokinin added to the cultured medium. Based on the
study conducted by Tawfik (1992), increasing the cytokinin (TDZ) in the
culture medium increased the camphor level and decreased the bornyl acetate
in the proliferated explant. Also, increasing BA in the regeneration medium
increased the camphor level and decreased the bornyllevel in the regenerant
plants. The changes in the proportion of the monoterpenes caused by TDZ
and BA suggested that the cytokinin may have an effect on the biosynthesis of
camphor. Since camphor can be derived from borneol (Croteau and Karp
1976), the cytokinin may affect the enzymes responsible for the conversion of
borneol or borneol acetate to camphor. Tawfik et a1. (1992a) reported that
increasing BA in the shoot-tip culture medium of Salvia officinalis L. increased camphor and decreased borneol. On the other hand, Drawert (1988)
reported the biotransformation of terpenes such as borneol to camphor and
citronellal to citonellol in cell suspension culture of Salvia officinalis and
Melissa sp., respectively. The rate and conversion time of one terpene to
another depended on the kind of monoterpene.

4 Summary
1. A protocol was established to induce organogenic callus of two genotypes
of rosemary (Rosmarinus officinalis) using thidiazuron (TDZ) alone or
with indoleacetic acid (IAA) at 0.5 mg/l.

364

A.A. Tawfik et al.

2. The use of IAA plus TDZ was essential to produce large masses of
organogenic callus from all types of explant used in the study.
3. The effect of TDZ varied depending upon the genotype.
4. Explants taken from the CV. Lockwood de Forest induced excellent callus,
but for Prostratus, shoot tips were the best explant to induce callus for plant
regeneration.
5. For shoot regeneration from callus, benzyladenine (BA) at 4mg/1 was the
best cytokinin treatment.
6. A significant effect of the plant growth regulators used in this study on the
monoterpenes identified in rosemary was observed.
7. Sucrose concentrations not only affected the growth of rosemary
cultures in vitro but also affected some oil constituents in both
genotypes.
8. Calcium chloride had a noticeable effect on the texture of the callus. Low
concentration of Ca2 + (O.99mMol) produced dark green and compact callus, while a higher concentration (S.99mMol/l) produced light green friable
callus.
9. Ca2 + ion significantly affected the oil yield and four of the monoterpenes
identified in the callus extract.

References
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muscle. J Ethnopharmacol 33:57-62
Bajaj YPS (1996) Biotechnology in agriculture and forestry, vol 37. Medicinal and aromatic plants
IX. Springer, Berlin Heidelberg New York
Banthorpe DV, Bilyard HO, Brown GD (1989) Enol esters of caffeic acid in serveral genera of
Labiatae. Phytochemistry 28:2109-2113
Browse PM (1986) Rosemaries. Pac-hortic. San Francisco: Pacific Horticultural Foundation (Fall
1986) 47:40-46
Calvo MC, Sanchez-Grass MC (1993) Accumulation of monoterpenes in shoot-proliferation
cultures of Lavandula latifolia Med. Plant Sci 91(2):207-212
Croteau R (1980) The biosynthesis of terpene compounds. In: Croteau R, Pattensen W
(eds) Fragrance and flavor substances. Springer Berlin Heidelberg New York, pp 13-36
Croteau R, Karp F (1976) Enzymatic synthesis of camphor from neryl pyrophosphate by
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Cuppett SL, Birt DF, Lawson T, Wheeler DDS, Hall CA (1992) Antioxidant, antimutagenic
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XIX Sesamum indicum L. (Sesame): In Vitro


Culture, and the Production of Naphthoquinone and
Other Secondary Metabolites

1 Introduction
1.1 Distribution and Importance of Sesame

The genus Sesamum (family Pedaliaceae) includes about 38 species, most


of these are wild types. S. indicum, which has been recognized as the only
cultivated species, comprises about 3000 varieties and strains. Although 30
species in the wild types are distributed in the savannas of Africa, S. indicum
has been cultivated in regions ranging from tropical to cold temperate zones
situated between 45 ON and 45 oS throughout the world (Kobayashi 1988,
1991).
S. indicum is an annual herb which grows to a height of approximately
100cm (Fig. 1), and may manifest varying morphological characteristics. For
example, S. indicum has the chromosome number 2n = 26, and other species
of sesame have 2n = 26, 32, 52, 58, 64.
The history of sesame cultivation is very old and it was recorded in
medicinal writings that sesame had already been cultivated in the Nile Basin
before 3000 B.C. (Nayar and Mehra 1970).
Sesame is one of the most important edible oil seed crops in the world, and
whole seeds may be eaten as a source of nutrition. It contains about 50% oil,
20-25% protein, 20% sugar, 6% fiber, and many kinds of minerals. The
frequency of germination in the white seed type was more than 80% even after
preservation for 30 years at normal temperature, and the oil contents did